ES2776657T3 - Self-buffering protein formulations - Google Patents

Abstract

A composition of a recombinant antibody and one or more pharmaceutically acceptable polyols, wherein at the pH of the composition, at 21 ° C, at one atmosphere and in equilibrium with the ambient atmosphere, the antibody has a buffer capacity per unit volume of at minus that of about 4.0 mM sodium acetate buffer in pure water in the range of pH 5.0 to 4.0 or pH 5.0 to 5.5 under the same conditions, and where further excluding the buffer capacity of said antibody, the buffer capacity per unit volume of the composition under the same conditions is not more than that of the 2.0 mM sodium acetate buffer in pure water in the range of pH 5.0 to 4.0 or pH 5.0 to 5.5 under the same conditions, where the antibody provides at least 80% of the buffer capacity of the composition, where the concentration of the antibody is between about 20 and 400 mg / ml, where the pH maintained by the buffering action of the antibody is between approximately 3.5 and 8.0, and wherein the antibody comprises: a heavy chain comprising the amino acid sequence of: ** (See sequence) ** and a light chain comprising the amino acid sequence of :: ** (See sequence) **

Description

DESCRIPTION

Self-buffering protein formulations

Field of the invention

The invention relates to the formulation of proteins, especially pharmaceutical proteins. In particular, it relates to self-buffering biopharmaceutical protein compositions, and to methods for designing, preparing, and using the compositions. It further relates to pharmaceutical protein compositions for veterinary and / or human medical use, and methods related thereto.

Background of the invention

Many aspects of pharmaceutical production and formulation processes are sensitive to pH. Maintaining the correct pH of a finished pharmaceutical product is critical to its stability, efficacy, and shelf life, and pH is an important consideration in designing formulations for administration that are acceptable as well as safe and effective.

To maintain pH, pharmaceutical formulations and processes use one or more buffering agents. A variety of buffering agents are available for pharmaceutical use. The buffer or buffers for a given application must be effective at the desired pH. They must also provide sufficient buffer capacity to maintain the desired pH for as long as necessary. A good buffer for a pharmaceutical composition must satisfy many other requirements as well. It must be adequately soluble. It must not form harmful complexes with metal ions, be toxic, or unduly penetrate, solubilize, or absorb onto membranes or other surfaces. It should not interact with other components of the composition in any way that diminishes its availability or effectiveness. It must be stable and effective in maintaining the pH within the range of conditions to which it will be exposed during formulation and during product storage. It should not be adversely affected by oxidation or other reactions that occur in its environment, such as those that occur in the processing of the composition in which it is providing the buffering action. If transferred or incorporated into a final product, a buffering agent must be safe for administration, compatible with other components of the composition during the shelf-life of the product, and acceptable for administration to the end user.

Although there are many general purpose buffers, only a limited number are suitable for biological applications, and of these, even fewer are acceptable for pharmaceutical formulations and processes. As a result, it is often difficult to find a buffer that is not only effective in maintaining pH, but also meets all the other requirements for a given pharmaceutical product, formulation or process.

The challenge of finding a suitable buffer for pharmaceutical use can be especially strong for pharmaceutical proteins. The conformation and activity of proteins are critically dependent on pH. Proteins are susceptible to a variety of pH-sensitive reactions that are detrimental to their efficacy, generally many more than small-molecule drugs. For example, to mention just a few prominent examples, asparagine and glutamine side chain amides deamidate at low pH (less than 4.0) and also at neutral or high pH (greater than 6.0). Aspartic acid residues promote hydrolysis of adjacent peptide bonds at low pH. The stability and arrangement of disulfide bonds are highly dependent on pH, particularly in the presence of thiols. The solubility, flocculation, aggregation, precipitation, and fibrillation of proteins are critically dependent on pH. The important crystal habit for some pharmaceutical formulations is also critically dependent on pH. And pH is also an important factor in the surface adsorption of many pharmaceutical peptides and proteins.

Furthermore, buffering agents that catalyze reactions that inactivate and / or degrade one or more other ingredients cannot be used in pharmaceutical formulations. Buffers for pharmaceutical use must not only have the necessary buffer capacity to maintain the correct pH, but also must not buffer so strongly that their administration detrimentally disturbs the physiological pH of a subject. Buffers for pharmaceutical formulations must also be compatible with generally complex formulation processes. For example, buffers that sublimate or evaporate, such as acetate and imidazole, generally cannot be used to maintain pH during lyophilization and in the reconstituted lyophilization product. Other buffers that crystallize in the amorphous phase of the protein, such as sodium phosphate, cannot be relied upon to maintain pH in processes that require freezing.

Buffers used to maintain pH in pharmaceutical end products must also be not only effective in maintaining pH, but also safe and acceptable for administration to the subject. For example, various otherwise useful buffers, such as low or high concentration citrate and high concentration acetate, are undesirably painful when administered parenterally.

Some buffers have been found to be useful in formulating pharmaceutical proteins, such as acetate, succinate, citrate, histidine (imidazole), phosphate, and Tris. They all have undesirable limitations and disadvantages. And everyone they have the inherent disadvantage of being an additional ingredient in the formulation, which complicates the formulation process, poses a risk of adversely affecting other ingredients, stability, shelf life and acceptability to the end user.

A need exists, therefore, for additional and improved methods of maintaining pH in the production and formulation of pharmaceuticals and in pharmaceutical compositions, particularly in the production and formulation of biopharmaceutical proteins and in biopharmaceutical protein compositions.

WO 03/002713 describes antibodies that interact with osteoprotegerin ligand (OPGL), methods of treating osteopenic disorders by administering a pharmaceutically effective amount of antibodies to OPGL, and methods of detecting the amount of OPGL in a sample using antibodies to OPGL.

The use of proteins as buffers is described in Halvor N. Christensen "Proteins as Buffers" (Annals of the New York Academy of Sciences, Vol. 133, No. 1 Current Conce, April 1, 1966, pages 34-30, XP055045656).

The influence of histidine on the stability and physical properties of a fully human antibody in aqueous and solid forms is described in Bei Chen et al ("Influence of histidine on the stability and physical properties of a fully human antibody in aqueous and solid forms ", Pharmaceutical Research, Kluwer Academic Publishers, New York, NY, US, vol. 20, no. 12, December 1, 2003, pages 1952-1960, XP002386671).

US 5,945,098 describes stable intravenously administrable immunoglobulin preparations stabilized against aggregation and polymerization and rendered isotonic with amino acids and with the non-ionic detergents, polysorbate and polyethylene glycol.

A specific molar ratio of stabilizer to protein is required for the storage stability of a lyophilized monoclonal antibody as described in Cleland et al (Cleland JL at al "a specific molar ratio of stabilizer to protein is required for storage stability of a lyophilized monoclonal antibody ", Journal of Pharmaceutical Sciences, American Pharmaceutical Association, Washington, US, vol. 90, no. 3, March 1, 2001, pages 310-321, XP001179875).

Summary

The present invention relates, in a first aspect, to a composition of a recombinant antibody and one or more pharmaceutically acceptable polyols, wherein at the pH of the composition, at 21 ° C, at an atmosphere and in equilibrium with the ambient atmosphere, The antibody has a buffering capacity per unit volume of at least that of about 4.0 mM sodium acetate buffer in pure water in the range of pH 5.0 to 4.0 or pH 5.0 to 5.5 under the same conditions, and where in addition, excluding the buffering capacity of said antibody, the buffering capacity per unit volume of the composition under the same conditions is not more than that of the 2.0 mM sodium acetate buffer in pure water in the range of pH 5.0 to 4.0 or pH 5.0 to 5.5 under the same conditions, where the antibody provides at least 80% of the buffer capacity of the composition, where the concentration of the antibody is between approximately 20 and 400 mg / ml, where the pH maintained by the buffer action The amount of the antibody is between about 3.5 and 8.0, and wherein the antibody comprises:

a heavy chain comprising the amino acid sequence of:

E VQLLESGGGL VQPGGSLRLS CAASGFTFSS YAMSWVRQAP GKGLEWVSGI

TGSGGSTYYA DSVKGRFTIS RDNSKNTLYL QMNSLRAEDT AVYYCAKDPG

TTVIMSWFDP WGQGTLVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLV

KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSW TVPSSNFGTQ

TYTCNVDHKP SNTKVDKTVE RKCCVECPPC PAPPVAGPSV FLFPPKPKDT

LMISRTPEVT CVWDVSHED PEVQFNWYVD GVEVHNAKTK PREEQFNSTF

R W S V L T W H QDWLNGKEYK CKVSNKGLPA PIEKTISKTK GQPREPQVYT

LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPMLDS

DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK;

and

a light chain comprising the amino acid sequence of:

EIVLTQSPGT LSLSPGERAT LSCRASQSVR GRYLAWYQQK PGQAPRLLIY

GASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVFYCQ QYGSSPRTFG

QGTKVEIKRT VAAPSVFIFP PSDEQLKSGT AS W CLLNNF YPREAKVQWK

VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ

GLSSPVTKSF NRGEC.

The pH maintained by the buffering action of the antibody can be between about 4 and 6.

The composition may further comprise one or more pharmaceutically acceptable salts. The total salt concentration can be less than 150 mM. The total salt concentration can be less than 100 mM. The polyol can be one or more of sorbitol, mannitol, sucrose, trehalose, or glycerol.

The composition may further comprise one or more pharmaceutically acceptable surfactants. The surfactant may be one or more of polysorbate 20, polysorbate 80, other sorbitan fatty acid esters, polyethoxylates, and poloxamer 188. The antibody may comprise a first binding moiety of a cognate pair of binding moieties. The antibody can be specific for OPGL.

The present invention also relates, in a second aspect, to a lyophilizate which after reconstitution provides a composition of the first aspect.

In a third aspect, the present invention relates to a kit comprising in one or more packages a composition of the first aspect and instructions regarding the use thereof or a lyophilizate of the second aspect and instructions regarding the use thereof.

The present invention also relates to a composition of the first aspect for use in medicine.

In a further aspect, the invention relates to a process for preparing a composition of the first aspect which comprises removing residual buffer using a counter ion or by diafiltration against a solution without buffer having a pH below the desired pH.

The present invention also relates in a further aspect to a process for preparing a composition of the first aspect which comprises removing residual buffer using any one or more of the following in the presence of a counter ion: size exclusion chromatography, dialysis and / or cross flow filtration and / or ion exchange chromatography.

Brief description of the figures

Figure 1 represents the titration data and the buffer capacity as a function of the concentration of standard sodium acetate buffers in the range of pH 5.0 to 4.0. Panel A is a graph depicting the change in pH after acid titration of several different concentrations of a standard sodium acetate buffer, as described in Example 1. pH is indicated on the vertical axis. The amount of acid added to each solution is indicated on the horizontal axis in microequivalents of HCl added per ml of solution (pEq / ml). Linear least squares trend lines are plotted for each data set. Acetate concentrations are indicated in the box. Panel B is a graph depicting the buffering capacity of acetate buffers over the acidic pH range as determined from the titration data plotted in Panel A, as described in Example 1. The buffering capacity is Indicates on the vertical axis as acid microequivalents per ml of buffer solution per unit of pH change (pEq / ml-pH). The acetate concentration is indicated on the horizontal axis in mM.

Figure 2 represents titration data and buffer capacity as a function of standard sodium acetate buffer concentrations in the range of pH 5.0 to 5.5. Panel A is a graph depicting the change in pH after base titration of several different concentrations of a standard sodium acetate buffer, as described in Example 2. pH is indicated on the vertical axis. The amount of base added to each solution is indicated on the horizontal axis in NaOH microequivalents added per ml of solution (pEq / ml). Linear least squares trend lines are plotted for each data set. Acetate concentrations are indicated in the box. Panel B is a graph depicting the buffering capacity of acetate buffers over the basic pH range as determined from the titration data plotted in Panel A and described in Example 2. The buffering capacity is indicated in the vertical axis as base microequivalents per ml of buffer solution per unit of pH change (pEq / ml-pH). The acetate concentration is indicated on the horizontal axis in mM.

Figure 3 represents the determination of the acetate concentration in acetate buffer standards, as described in Example 3. The graph shows a standard curve for the determinations, with the maximum area indicated on the vertical axis and the acetate concentration indicated on the horizontal axis. The nominal amounts and the measurements of acetate in the solutions used for the empirical determination of the buffer capacity are tabulated below the graph.

Figure 4 is a graph depicting the change in pH after acid titration of several different concentrations of Ac-hOPGL in the range of pH 5.0 to 4.0, as described in Example 4. The pH is indicated in the vertical axis. The amount of acid added to the solutions is indicated on the horizontal axis in HCl microequivalents added per ml of solution (pEq / ml). Linear least squares trend lines are plotted for each data set. Ac-hOPGL concentrations are indicated in the box.

Figure 5 is a graph depicting the change in pH after base titration of several different concentrations of Ac-hOPGL in the range of pH 5.0 to 6.0, as described in Example 5. The pH is indicated in the vertical axis. The amount of base added to solutions is indicated on the horizontal axis in microequivalents of NaOH added per ml of solution (pEq / ml). Linear least squares trend lines are plotted for each data set. Ac-hOPGL concentrations are indicated in the box.

Figure 6 shows the residual acetate levels in the Ac-hOPGL solutions used to determine the buffer capacity. The graph shows the standard curve used for the acetate determinations as described in Example 6. Nominal and experimentally measured acetate concentrations in the solutions are tabulated below the graph.

Figure 7 is a graph representing the Ac-hOPGL buffer capacity plus or minus residual acetate in the pH range 5.0 to 4.0. The data was obtained as described in Example 7. The upper line shows the buffering capacity of Ac-hOPGL with residual acetate. The bottom line shows the AchOPGL buffer capacity adjusted for residual acetate. The vertical axis indicates the buffer capacity in acid microequivalents per ml of Ac-hOPGL solution per pH unit (pEq / ml-pH). The horizontal axis indicates the AchOPGL concentration in mg / ml. The buffer capacities of different concentrations of standard acetate buffers as described in Example 1 are shown as horizontal lines. The concentrations of the buffers are indicated on the lines.

Figure 8 is a graph representing the Ac-hOPGL buffer capacity plus or minus residual acetate in the basic pH range of 5.0 to 6.0. Data were obtained as described in Example 8. The top line represents the buffering capacity of Ac-hOPGL with residual acetate. The bottom line represents the Ac-hOPGL buffer capacity adjusted for residual acetate. The vertical axis indicates the buffer capacity in added base microequivalents per ml of buffer solution per pH unit (pEq / ml-pH). The horizontal axis indicates the Ac-hOPGL concentration in mg / ml. The buffer capacities of different concentrations of standard sodium acetate buffers as described in Example 2 are indicated by horizontal lines. Acetate concentrations are indicated on each line.

Figure 9 represents, in a pair of graphs, the pH and stability of Ac-hOPGL in self-buffering and conventionally buffered formulations. Panel A represents the stability of self-buffered Ac-hOPGL, Ac-hOPGL formulated in acetate buffer, and Ac-hOPGL formulated in glutamate as a function of storage time at 4 ° C for a period of six months. The vertical axis indicates the stability of Ac-hOPGL in percentage of Ac-hOPGL monomers determined by SE-HPLC. Acetate storage time is indicated on the horizontal axis. Panel B represents the pH of the same three formulations measured during the same period of time. Protein stability determinations and pH measurements are described in Example 9. Figure 10 represents the titration curves and buffer capacities for various concentrations of self-buffering Ac-hB7RP1 formulations in the pH 5.0 range. to 4.0. Panel A shows the titration data. The pH is indicated on the vertical axis. The amount of acid added to the solutions is indicated on the horizontal axis in HCl microequivalents added per ml of solution (pEq / ml). Linear least squares trend lines are plotted for each data set. The hB7RP1 concentrations are indicated in the box. Panel B represents the buffer capacities of the Ac-hB7RP1 formulations. The top line shows the buffer capacities for the formulations, including the contribution of residual acetate. The bottom line shows the buffer capacities for formulations after subtracting the residual acetate contribution based on SE-HPLC determinations as described in Example 3. Linear least squares trend lines are shown for the two data sets. The vertical axis indicates the buffer capacity in acid microequivalents per ml of buffer solution per pH unit (pEq / ml-pH). AchB7RPl concentration is indicated on the horizontal axis in mg / ml. The buffer capacities of different concentrations of standard sodium acetate buffers as described in Example 1 are shown with dashed horizontal lines. The acetate buffer concentration is shown below each line. Data was obtained as described in Reference Example 1.

Figure 11 depicts the titration curves and buffer capacities for various concentrations of self-buffering Ac-hB7RP1 formulations in the pH range 5.0 to 6.0. Panel A shows the titration data. The pH is indicated on the vertical axis. The amount of base added to solutions is indicated on the horizontal axis in microequivalents of NaOH added per ml of solution (pEq / ml). Linear least squares trend lines are plotted for each data set. The hB7RP1 concentrations are indicated in the box. Panel B represents the buffer capacities of the Ac-hB7RP1 formulations. The top line shows the buffer capacities for formulations containing residual acetate. The bottom line shows the buffer capacities for formulations adjusted to eliminate the contribution of residual acetate. Linear least squares trend lines are shown for the two data sets. The vertical axis indicates the buffer capacity in base microequivalents per ml of buffer solution per pH unit (pEq / mlpH). The concentration of Ac-hB7RP1 is indicated on the horizontal axis in mg / ml. The buffer capacities of Different concentrations of standard sodium acetate buffers as described in Example 2 are shown with dashed horizontal lines. Acetate buffer concentrations are shown below each line. Data was obtained as described in Reference Example 2.

Figure 12 depicts the stability of Ac-hB7RP1 in self-buffering and conventionally buffered formulations at 4 ° C and 29 ° C. Panel A represents the stability of self-buffered Ac-hB7RP1, Ac-hB7RP1 formulated in acetate buffer and Ac-hB7RP1 formulated in glutamate as a function of storage at 4 ° C for a period of six months. The vertical axis represents the Ac-hB7RP1 monomer in the samples determined by SE-HPLC. Time is indicated on the horizontal axis. Panel B represents the stability of the same three formulations as a function of storage at 29 ° C for the same period of time. The axes in Panel B are the same as in Panel A. Determinations of protein stability by HPLC-SE are described in Reference Example 3.

Figure 13 represents the pH stability of Ac-hB7RP1 autbuffer formulations at 4 ° C and 29 ° C. The vertical axis indicates pH. The time, e weeks, is indicated on the horizontal axis. The temperatures of the data sets are indicated in the box. Data was obtained as described in Reference Example 4.

Figure 14 represents the buffering capacity of the Ac-hCD22 self-buffering formulations as a function of the Ac-hCD22 concentration in the range of pH 4.0 to 6.0. Panel A represents the buffering capacities of the self-buffering Ac-hCD22 formulations as a function of the Ac-hCD22 concentration in the range of pH 4.0 to 5.0. Panel B represents the buffer capacities of the self-buffering Ac-hCD22 formulations as a function of concentration in the range of pH 5.0 to 6.0. In both panels, the vertical axis indicates the buffer capacity in base microequivalents per ml of buffer solution per pH unit (pEq / ml-pH) and the horizontal axis indicates the Ac-hCD22 concentrations in mg / ml. For reference, the 10 mM sodium acetate buffer capacity as described in Example 1 is shown in both panels by a horizontal dashed line. The results shown in the Figure were obtained as described in Reference Example 5.

Figure 15 depicts the titration curves and buffer capacities for various concentrations of self-buffering Ac-hIL4R formulations in the range of pH 5.0 to 4.0. Panel A shows the titration data. The pH is indicated on the vertical axis. The amount of acid added to the solutions is indicated on the horizontal axis in HCl microequivalents added per ml of solution (pEq / ml). Linear least squares trend lines are plotted for each data set. The hIL4R concentrations are indicated in the box. Panel B represents the Ac-hIL4R buffer capacities as a function of concentration. The least squares linear trend line is shown for the data set. The vertical axis indicates the buffer capacity in base microequivalents per ml of buffer solution per pH unit (pEq / ml-pH). The Ac-hIL4R concentration is indicated on the horizontal axis in mg / ml. The buffer capacities of different concentrations of standard sodium acetate buffers as described in Example 1 are shown with dashed horizontal lines. Acetate buffer concentrations are shown below each line. Data was obtained as described in Reference Example 6.

Figure 16 depicts the titration curves and buffer capacities for various concentrations of self-buffering Ac-hIL4R formulations in the range of pH 5.0 to 6.0. Panel A shows the titration data. The pH is indicated on the vertical axis. The amount of base added to solutions is indicated on the horizontal axis in microequivalents of NaOH added per ml of solution (pEq / ml). Linear least squares trend lines are plotted for each data set. The hIL4R concentrations are indicated in the box. Panel B represents the Ac-hIL4R buffer capacities as a function of concentration. The least squares linear trend line is shown for the data set. The vertical axis indicates the buffer capacity in base microequivalents per ml of buffer solution per pH unit (pEq / ml-pH). The Ac-hIL4R concentration is indicated on the horizontal axis in mg / ml. The buffer capacities of different concentrations of standard sodium acetate buffers as described in Example 2 are shown with dashed horizontal lines. Acetate buffer concentrations are shown below each line. Data was obtained as described in Reference Example 7.

Figure 17 depicts Ac-hIL4R and pH stability in acetate buffered and autotamped formulations of Ac-hIL4R at 37 ° C as a function of time. Panel A is a bar graph showing the stability of Ac-hIL4R for four weeks at 37 ° C. The vertical axis indicates stability in percent of monomeric AchIL4R as determined by SE-HPLC. The horizontal axis indicates the storage time in weeks. The insert identifies the data for the acetate and for the self-buffered formulations. Panel B shows the pH stability of the same formulations for the same conditions and time periods. The pH is indicated on the vertical axis. Storage time in weeks is indicated on the horizontal axis. Data for acetate and self-buffered formulations are in the box. Data was obtained as described in Reference Example 8.

Glossary

The meanings attributed to different terms and phrases as used herein are explained illustratively below.

"One" or "one" herein means "at least one"; "one or more than one".

"About", unless otherwise explicitly stated herein, means V 20%. For example, about 100 herein means from 80 to 120, about 5 means from 4 to 6, about 0.3 means 0.24 to 0.36 and about 60 % means 48 % to 72 % (not 40 % to 80%).

"Agonist (s)" means herein a molecular entity that is different from a corresponding stimulating ligand but has the same stimulating effect. For example (although agonists work through other mechanisms), for a hormone that stimulates an activity by binding to a corresponding hormone receptor, an agonist is a chemically different entity that binds to the hormone receptor and stimulates its activity.

"Antagonist (s)" means herein a molecular entity that is different from a corresponding stimulatory ligand and has an opposite effect. For example (although antagonists work through other mechanisms), a type of antagonist of a hormone that stimulates an activity by binding to a corresponding hormone receptor is a chemical entity that is different from the hormone and binds to the hormone receptor but does not stimulates the activity generated by the binding of the hormone, and through this action inhibits the effector activity of the hormone.

"Antibody (s)" is used herein in accordance with its usual meaning in biochemical and biotechnological techniques.

Antibodies within the meaning of the term as used herein include isolates from biological sources, including monoclonal and polyclonal antibodies, antibodies prepared by recombinant DNA techniques (also sometimes referred to herein as , recombinant antibodies), including those prepared by processes that involve the activation of an endogenous gene and those that involve the expression of an exogenous expression construct, including antibodies prepared in cell culture and those prepared in transgenic plants and animals, and antibodies prepared by methods involving chemical synthesis, including peptide synthesis and semi-synthesis. Also within the scope of the term as used herein, unless explicitly stated otherwise, are chimeric antibodies and hybrid antibodies, among others.

The prototypic antibody is a tetrameric glycoprotein composed of two identical heavy chain-light chain dimers linked together by disulfide bonds. There are two types of light chains in vertebrates, kappa and lambda. Each light chain comprises a constant region and a variable region. The two light chains are distinguished by constant region sequences. There are five types of heavy chains in vertebrates: alpha, delta, epsilon, gamma, and mu. Each heavy chain comprises a variable region and three constant regions. The five types of heavy chain define five classes of vertebrate antibodies (isotypes): IgA, IgD, IgE, IgG, and IgM. Each isotype is composed of, respectively, (a) two alpha, delta, epsilon, gamma, or mu heavy chains, and (b) two kappa or two lambda light chains. The heavy chains in each class associate with both types of light chains; But, the two light chains in a given molecule are both kappa or both lambda. IgD, IgE and IgG generally occur as "free" heterotetrameric glycoproteins. IgA and IgM generally occur in complexes comprising heterotetramers of various IgA or various IgM associated with a "J" chain polypeptide. Some isotypes of vertebrates are classified into subclasses, which are distinguished from each other by differences in constant region sequences. There are four subclasses of human IgG, IgG1, IgG2, IgG3, and IgG4, and two subclasses of IgA, IgA1 and IgA2, for example. All of these and others not specifically described above are included within the meaning of the term "antibody (s)" as used herein.

The term "antibody (s)" further includes amino acid sequence variants of any of the foregoing as further described elsewhere herein.

"Antibody derivative" as used herein means any protein produced from an antibody, and any protein of an antibody-based design. The term includes within its meaning proteins produced using all or part of an antibody, those that comprise all or part of an antibody, and those designed in whole or in part based on all or part of an antibody. Proteins "derived from antibodies" include, but are not limited to, Fc, Fab and Fab2 fragments and proteins comprising the same, V ^h domain and V ^l domain fragments and proteins comprising the same, other proteins comprising a variable region and / or an antibody constant, in whole or in part, intrabodies, maxibodies, minibodies, scFv (s) diabodies, amino acid sequence variants of the foregoing, and a variety of other such molecules, including but without limitation to others described elsewhere herein.

"Antibody related", as used herein, means any protein or mimetic that resembles in structure, function, or design an antibody or any part of an antibody. Among the "antibody-related" proteins as the term is used herein are the "antibody-derived" proteins as described above. It should be noted that the terms "derived from antibodies" and "related to antibodies" substantially overlap; both terms apply to many of those proteins. Examples of "antibody related" proteins, without implying limitation in this regard, are peptibodies and receptors. Other examples of "antibody related" proteins are described elsewhere herein.

"Antibody polypeptide (s)" as used herein, except where otherwise indicated, means a polypeptide that is part of an antibody, such as a light chain polypeptide, a heavy chain polypeptide, and a polypeptide of J chain, to mention a few examples, including among other fragments, derivatives and variants thereof, and related polypeptides.

"Around" unless otherwise stated means the same as about.

"Binding residue (s)" means a part of a molecule or a complex of molecules that specifically binds part of another molecule or complex of molecules. The binding moiety can be the same or different from the part of the molecule or complex of molecules to which it is bound. The linking moiety can also be an entire molecule or complex of molecules.

"Binds specifically" is used herein in accordance with its usual meaning in the arts and means, unless otherwise indicated, that the binding is stronger with certain specific moieties than with other moieties in general, that it is more stronger than the nonspecific binding that can occur with a wide variety of residues, and that the binding is selective for certain residues and does not occur as strongly with others. In the extreme case of specific binding, there is a very strong binding with only one type of residue, and there is no non-specific binding with any other residue.

"Co-administering" means an administration of two or more agents in conjunction with one another, including simultaneous and / or sequential administration.

"Affine or related" in this document means complementary, matching, coincident, such as, for example, two matching puzzles, the mechanism of a lock cylinder and the key that opens it, the attachment site of the substrate of an enzyme and the substrate of the enzyme and a target and a target-binding protein that specifically binds to it.

"Cognate binding moieties" as used herein means binding moieties that specifically bind to each other. Generally, but not always, it means a pair of linking moieties that specifically bind to each other. The moieties responsible for the highly selective binding of a specific ligand and ligand receptor provide an illustrative example of cognate binding moieties. Another example is provided by moieties that bind an antigen and an antibody.

"Composition" means any composition of matter comprising one or more constituents, such as a formulation.

"Composed by is synonymous with" comprising "(see below).

"Comprising" means that it includes, without further qualification, limitation or exclusion as to what else may or may not be included. For example, "a composition that comprises x and y" means any composition that contains x and y, no matter what else it may contain. Similarly, "a method comprising x" is any method in which x is performed, no matter what else may be produced.

"Concentration" is used herein according to its well-known meaning in the art to indicate the amount of an article in a given quantity of a mixture containing the article, generally expressed as a ratio. For example, the concentration of a solute, such as a protein in a solution, can be expressed in many ways, such as (but not limited to: (A) Weight percent (i) = weight of solute per 100 volume units of solvent; (B) Percentage by weight (ii) = weight of solute per 100 units of total weight; (C) Percentage by weight (iii) = weight of solute per 100 units of solvent by weight; (D) Percentage by mass = mass of solute per 100 mass units of solution; (E) Molar fraction = moles of solute per total moles of all components; (F) Molarity = moles of solute per liter of solution (i.e., solute plus solvent); ( G) Molality = moles of solute per kg of solvent; and (H) Molality in volume = moles of solute per liter of solvent.

"Control region (s)" is used herein in accordance with its meaning well known in the art, and unless otherwise indicated, refers to regions in DNA or proteins that are responsible for controlling one or more functions or activities thereof. For example, "expression control region" with reference to the control of gene expression, means the regions in DNA that are needed for transcription to occur correctly and that are involved in the regulation of when transcription occurs, how efficiently it is produced, when it stops, and the like.

"De novo" is used herein in accordance with its usual meaning in the art, to denote something done anew. For example, a de novo amino acid sequence is one that is not derived from a naturally occurring amino acid sequence, although such a de novo sequence may have similarities to a naturally occurring sequence. De novo amino acid sequences can be generated, for example, by a priori design , by combinatorial methods, by selection methods. They can be prepared, for example, by chemical synthesis, by synthesis, and by a variety of recombinant DNA techniques, all of which are well known to those of skill in the art.

"Harmful" means, as used herein, harmful. By way of illustration, "harmful" processes include, for example, harmful effects of disease processes and harmful side effects of treatments.

"Derivative (s)" is used herein to denote derived from, in substance, form, or design, such as, for example, a polypeptide that is based on but differs from a reference polypeptide, for example, by alterations in its amino acid sequence, by fusion to another polypeptide, or by covalent modification.

"Disease (s)", a pathology, a condition that adversely affects the health of a subject.

"Disorder (s)" a threat, a condition that adversely affects health. "Dysfunction" means, as used herein, a disorder, disease, or detrimental effect of a normal process.

"Effective amount" generally means an amount that provides the desired local or systemic effect. For example, an effective amount is an amount sufficient to achieve a beneficial or desired clinical result. The effective amount can be provided all at once in a single administration or in fractional amounts that provide the effective amount in multiple administrations. The precise determination of what would be considered an effective amount can be based on individual factors for each subject, including their size, age, injury and / or disease or injury to be treated and the amount of time since the injury has occurred or has begun. the illness. One skilled in the art will also be able to determine the effective amount for a given subject based on these considerations that are common in the art. As used herein, "effective dose" means the same as "effective amount."

"Effective route" generally means a route that provides delivery of an agent to a desired compartment, system, or location. For example, an effective route is one through which an agent can be administered to provide at the desired site of action an amount of the agent sufficient to effect a beneficial or desired clinical result.

"Endogenous" (such as endogenous gene) is used herein to refer to, for example, genes and other aspects of DNA, such as control regions, that are naturally occurring in a genome and organism, unless stated indicate otherwise.

Exogenous "(such as exogenous gene), unless otherwise indicated, is used herein generally to denote, for example, DNA from an external source, such as DNA introduced into a cell and incorporated into its genome.

"FBS" means fetal bovine serum.

"Formulation (s)" means a combination of at least one active ingredient with one or more other ingredients for one or more particular uses, such as storage, further processing, sale, and / or administration to a subject, such as, for example, administration to a subject of a specific agent in a specific amount, by a specific route, to treat a specific disease.

"Fragment (s)" herein means part of a larger entity, such as a part of a protein; for example, a polypeptide that consists of less than the complete amino acid sequence of a larger polypeptide. As used herein, the term includes fragments formed by terminal deletion and fragments formed by internal deletion, including those in which two or more non-contiguous portions of a polypeptide are joined together to form a smaller polypeptide, which is a fragment of the original.

"Fusion protein (s)" herein means a protein formed by fusing all or part of two polypeptides, which may be the same or different. Typical fusion proteins are produced by recombinant DNA techniques, by end-to-end joining of nucleotides encoding the two (or more) polypeptides.

"Genetically engineered" as used herein means produced using an intentional genetic alteration process, such as by recombinant DNA technology, classical genetic engineering methods, chemical methods, a combination of the three, or other methods.

"Homologous (s)" herein means having homology to another entity, such as a protein that is homologous to another protein. Homologous means to resemble each other in structure or function.

"Ionization" herein means the change of the net charge on a substance by at least one, including the loss or gain of charge, such as ionization of acetic acid in low pH solution, from HOAc to OAc- and H + .

"k" herein denotes an equilibrium coefficient, in accordance with its conventional meaning in chemistry.

"ka" herein denotes the dissociation constant of a particular hydrogen from a molecule, in accordance with its conventional meaning in chemistry, such as, for example, the dissociation constant of acidic hydrogen from acetic acid.

"kd" herein denotes a dissociation constant of a pair of chemical entities (or moieties), in accordance with its conventional meaning in chemistry.

Kit "means a collection of items used together for a given purpose or purposes.

"Ligand (s)" herein means a molecular entity that selectively and stoichiometrically binds to one or more specific sites on different other molecular entities. The bond is generally not covalent, but it can also be covalent. A few examples, among many others, are (a) antigens, which generally bind non-covalently to binding sites on cognate antibodies; (b) hormones, which generally bind to hormone receptors, non-covalently; (c) lectins, which bind specific sugars, non-covalently; (d) biotins, which bind to multiple sites on avidin and other avidin-like proteins, non-covalently; (e) hormonal antagonists, which bind to hormone receptors and inhibit their activity and / or that of the corresponding hormone; and (f) hormone agonists, which bind similarly to hormone receptors but stimulate their activity.

"Ligand binding moiety (s)" herein means a molecular entity that binds to a ligand, generally a part of a larger molecular entity that binds to the ligand, or a molecular entity derived therefrom.

"Ligand-binding protein (s)" herein means a protein that binds to a ligand.

"Ligand residue (s)" herein means a molecular entity that binds to a ligand-binding molecular entity in the same manner as the corresponding ligand. A ligand moiety can be all or part of a ligand, derived from a ligand, or generated de novo. Generally, however, the ligand moiety is more or less exclusively the element thereof that binds to the corresponding ligand binding entities. The ligand moiety need not comprise, and the term generally does not denote, structural features other than those necessary for ligand binding.

"mEq" herein means milliequivalent (s).

"| jEq" herein means microequivalent (s).

"Mimetic (s)" herein means a chemical entity with structural or functional characteristics of another, generally unrelated chemical entity. For example, one type of hormone mimetic is a non-peptide organic molecule that binds to the corresponding receptor in the same way as the corresponding hormone.

"mM" means millimolar; 10-3 moles per liter.

"Modified protein (s)", "modified polypeptide (s)", or "modified fragment (s)" herein means a protein or a polypeptide or a fragment of a protein or polypeptide comprising a chemical residue (structure) other than those of the twenty naturally occurring amino acids that form naturally occurring proteins. Modifications are most often covalently attached, but can also be non-covalently attached to a protein or other polypeptide, such as a fragment of a protein.

"Rest (s)" herein means a molecular entity that represents a specific structure and / or function, without foreign components. For example, in most cases, only a small part of a ligand binding protein is responsible for ligand binding. This part of the protein, whether encoded continuously or discontinuously, is an example of a ligand-binding moiety.

"Of natural origin" means in nature, without human intervention.

"Non-naturally occurring" means that it does not occur in nature or, if it occurs in nature, is not in its naturally occurring state, environment, circumstances or the like.

"PBS" means phosphate buffered saline.

"Peptibody" refers to a molecule comprising an antibody Fc domain (ie, C ^h 2 and C ^h 3 antibody domains) which excludes the C ^h 1, C ^l , V ^h, and V ^l antibody domains, thus as Fab and F (ab) 2, where the Fc domain is linked to one or more peptides, preferably to a pharmacologically active peptide, particularly preferably to a randomly generated pharmacologically active peptide. Peptibody production is generally described in PCT publication WO00 / 24782, published May 4, 2000, particularly regarding the structure, synthesis, properties and uses of peptibodies.

"Peptide (s)" herein means the same as polypeptide; often, but not necessarily, it is used in reference to a relatively short polypeptide,

"pH" is used according to its well known and universal definition as follows:

pH = - log [H3O +].

"Pharmaceutical product", as used herein, means that it is acceptable for use in a human or non-human subject for the treatment thereof, particularly for use in humans, and approved therefor by an empowered regulatory authority. to regulate the use thereof such as, for example, the Food and Drug Administration in the United States, the European Agency for the Evaluation of Medicinal Products, the Japan's Ministry of Health, Labor and Welfare, or other regulatory agency such as those listed in R. Ng, DRUGS: FROM DISCOVERY TO APPROVAL, Wiley-Liss (Hoboken, NJ) (2004) particularly regarding regulatory authorities related to drug approval, especially those listed in Chapter 7. As used herein document, the phrase "where the composition has been approved for pharmaceutical use by an authority legally empowered to grant said approval" means an entity or institution or similar, established by law and charged by law with the responsibility and power to regulate and approve the use of drugs for use in humans, and in some cases, non-humans. The approval of any of these agencies anywhere meets this qualification. The approving agency does not have to be that of the state in which, for example, a violation is being committed. Examples of such entities include the US Food and Drug Administration and the other agencies listed herein above.

As used herein, "pharmaceutical product" can also refer to a product produced in accordance with good manufacturing practices, such as those described in, among others, Chapter 9 and Chapter 10, of R. Ng, DRUGS: FROM DISCOVERY TO APPROVAL, Wiley-Liss (Hoboken, NJ) (2004) particularly in the parts pertinent to good manufacturing practices for pharmaceutical protein formulations, in particular, as set out in Chapters 9 and 10.

"Pharmaceutically acceptable" is used herein according to its meaning well known in the art to denote what is acceptable for medical or veterinary use, preferably for human medical use, particularly approved for such use by the Food and Drug. US Administration or other authority as described above with respect to the meaning of "pharmaceutical product".

"Polypeptide (s)" see "Protein (s)".

"Precursor (s)" is used herein in accordance with its well-known meaning in the art to denote an entity from which another entity is derived. For example, a precursor protein is a protein that undergoes processing, such as proteolytic cleavage or modification, thereby resulting in another precursor protein (which will undergo further processing) or a mature protein.

"Protein (s)" herein means a polypeptide or a complex of polypeptides, in accordance with its meaning well known in the art. As used herein, "protein (s)" includes both straight chain and branched polypeptides. It includes unmodified and modified polypeptides, including naturally occurring and non-naturally occurring modifications. Such modifications include chemical modifications of the ends, of the peptide backbone, and amino acid side chains; amino acid substitutions, deletions and additions; and incorporation of unusual amino acids and other residues, to name just a few of those modifications. It also includes "engineered" polypeptides and complexes thereof, such as, but not limited to, any polypeptide or polypeptide complex that has been intentionally altered in structure by, for example, recombinant DNA techniques, chemical synthesis, and / or covalent modification, including intentional alteration of the amino acid sequence and / or post-translational modifications.

"Protonation" means the addition of at least one hydrogen.

"Self-buffering" means the ability of a substance, such as a pharmaceutical protein, to withstand the change in pH sufficient for a given application, in the absence of other buffers.

"Semi-de novo" herein means (a) partially designed in accordance with a particular reference and / or produced from a precursor, and (b) partially designed without reference to a particular reference (such as designed solely by general principles and not based on any particular reference). For example, a polypeptide made by producing a first peptide in a bacterial expression system, producing a second peptide by chemical synthesis, and then linking the two peptides together to form the polypeptide.

"Semisynthesis" means, as used herein, a combination of chemical and non-chemical synthetic methods.

"Subject" means a vertebrate, such as a mammal, such as a human. Mammals include, but are not limited to, humans, farm animals, sport animals, and pets. Subjects in need of treatment by methods and / or compositions of the present invention include those suffering from a disorder, dysfunction or disease, or a side effect thereof, or a side effect of a treatment thereof.

"Substantially" is used herein according to its plain and ordinary definition meaning to a great extent or degree. For example, substantially complete means to a great extent complete, to a great extent complete. By way of further illustration, "substantially free of debris" means largely free of debris, largely free. If numerical precision is required, depending on the context, "substantially" as used herein means at least 80% or more, particularly 90% or more, very particularly 95% or more.

"Therapeutically effective" is used herein in accordance with its well-known meaning in the art to denote that which achieves an improvement in the prognosis or condition of a subject or which otherwise achieves a therapeutic goal, including, for example , a reduction in the rate of progression of a disease, even if a subject's condition nevertheless continues to deteriorate.

"Therapeutically effective amount" is generally used to qualify the amount of an agent to encompass those amounts that achieve an improvement in the severity of the disorder. For example, effective neoplastic therapeutics prolong the survivability of the subject, inhibit rapidly proliferating cell growth associated with neoplasia, or effect regression of the neoplasia. Treatments that are therapeutically effective within the meaning of the term as used herein include treatments that improve the quality of life of a subject even if they do not improve the outcome of the disease per se.

"Treat" that treats "or" treatment "are widely used in connection with the invention and each of these terms encompasses, but is not limited to, prevention, amelioration, inhibition, or cure of a deficiency, dysfunction, disease, or other deleterious process, including the that interfere with and / or are the result of therapy.

"Variant (s)" herein means a naturally-occurring or synthetic version of, for example, a protein that is structurally different from the original but is related in structure and / or function, such as an allelic variant, a paralog or a homologue of a protein.

Description of the invention

The invention provides for the first time self-buffering protein formulations, particularly biopharmaceutical protein formulations, methods of preparing the formulations, and methods of using the formulations, among other things. Any protein that provides sufficient buffering capacity within the necessary pH range at a concentration suitable for its intended use can be prepared as a self-buffering protein formulation in accordance with the invention. The invention can be practiced with a variety of proteins, including both naturally occurring proteins and "genetically engineered" proteins, particularly biopharmaceutical proteins, as discussed below.

The utility of proteins, particularly biopharmaceutical proteins, for formulating into self-buffering compositions, particularly pharmaceutically acceptable compositions, had not been recognized prior to the invention disclosed herein. The influence of proteins on the regulation of physiological pH has been recognized and studied for some time. However, until now it has not been recognized that proteins, particularly biopharmaceutical proteins, can have sufficient buffering capacity to maintain a formulation within a desired pH range, without additional buffering agents.

Biopharmaceutical proteins for use in the United States are formulated as buffered solutions, non-buffered solutions, amorphous or crystalline suspensions, and lyophilisates.

Most buffer solution formulations use a conventional buffering agent. Two proteins, Pulmozyme® and Humulin®, are formulated as solutions without conventional buffering agents. Neither of these proteins provide substantial self-buffering capacity in formulations.

Pulmozyme® has a molecular weight of approximately 37,000 Daltons and contains 5 histidines, 22 aspartic acids and 12 glutamic acids, among its 260 amino acids. The buffering capacity of the protein within 0.5 pH units of pH 6.3 is substantially determined by its histidine content. Based on this, the upper limit of the self-buffering capacity of the formulation is determined by the effective concentration of the histidine residues, 0.15 mM. The molar concentration of aspartic acid and glutamic acid in the formation is 0.9 mM. The total molar concentration of the three amino acids together, therefore, is slightly more than 1 mM, at the concentration of the formulation.

Humulin® is formulated at 3.5 g / ml. It has a molecular weight of approximately 6,000 Daltons and contains 2 aspartic acids, 8 glutamic acids, and 2 histidines. Neither of these amino acids is a particularly effective buffer at formulation pH: 7.0 to 7.8. At this concentration, the molar concentration of histidines, which are closest in pKa to the pH of the formulation, is 1.16 mM.

Biopharmaceutical lyophilisates are reconstituted before use into solutions or suspensions. Most lyophilizates contain conventional buffers that maintain the proper pH of reconstituted formulations. Some others, in which the protein concentration is low or the pH must be low (less than 3) or high (greater than 9.5), are not buffered effectively.

Therefore, buffering is achieved in current biopharmaceutical protein formulations using conventional buffering agents. The ability of proteins by themselves to buffer pharmaceutical protein formulations has not been fully appreciated and has not been used for the manufacture of protein pharmaceuticals.

Determination of protein buffering capacity is generally important in developing self-buffering protein formulations in accordance with the invention. Related to this, the methods for measuring the buffering capacity and for determining the buffering capacity of proteins are described below. To allow easy comparability of the data, the buffering capacity of proteins should be expressed in comparable units and / or related to a buffer standard. Accordingly, the following section describes the pH metrics and standards that meet these requirements, in accordance with the invention.

1. Buffering

A widely accepted definition of buffering is the resistance to change in the pH of a composition upon addition of acid or base. Buffer capacity, therefore, is often defined as the ability of a composition to resist change in pH.

Generally, buffering capacity is expressed in terms of the amount of strong acid or base necessary to change the pH of a composition by a given amount. Van Slyke provided the most widely used quantitative measure of buffering capacity, according to which, for a solution, the buffering capacity is expressed as the amount of strong acid or base required to change the pH of one liter of the solution by one unit of pH under natural conditions of temperature and pressure.

According to this measurement, for example, the buffer capacity of 1 liter of 5 mM HOAc, 5 mM NaOAc, pH 4.76 in pure water is 4.09 x 10-3 moles of a strong univalent base (i.e. 4 , 09 x 10-3 base equivalents), which can be calculated as follows.

The Henderson-Hasselbalch equation for the solution is:

pH = log {NaOAc [5 mM] / HOAc [5 mM]} 4.76

Therefore, the concentration, X, of a strong univalent base required to increase the pH of this buffer is:

4.76 to 5.76 is 5.76 = log {NaOAc [5 mM X mM] / HOAc [5 mM-X mM]} 4.76

Thus:

1.00 = log {NaOAc [5 mM X mM] / HOAc [5 mM-X mM]}

10.0 = NaOAc [5 mM X mM] / HOAc [5 mM - X mM]

10.0 = (5 mM X mM) / (5 mM - X mM)

50 mM - 10X mM = 5 mM XmM

11X mM = 45 mM

X = 4.09 mM,

that, for yields of one liter:

(4.09 x 10.3 moles / liter) (1 liter) (l equivalent / mole) = 4.09 x 10.3 equivalents

Therefore, according to this measurement, the buffer capacity of 1 liter of a 10 mM acetate buffer containing 5 mM NaOAc and 5 mM HOAC at a pH of 4.76 in pure water is 4.09 x 10'3 base equivalents per liter per pH unit. In other words, the buffer capacity of the solution is 4.09 milliequivalents of base per liter per pH unit, 4.09 microequivalents of base per milliliter per pH unit, 0.409 microequivalents of base per 100 microliters per pH unit , 40.9 nanomoles of base per 10 microliters per pH unit, and 4.09 nanomoles of base per microliter per pH unit.

The same calculation produces the following buffer capacity for other concentrations of this acetate buffer at pH 4.76. A 2 mM acetate buffer as above has a buffer capacity of 0.818 mEq per liter per pH unit. At 4 mM, the buffer capacity is 1.636 mEq per liter per pH unit. The capacity at 5 mM is 2,045 mEq per liter per pH unit. At 7.5 mM, the capacity is 3.068 mEq per liter per pH unit. At 10 mM, the acetate buffer has a buffer capacity of 4,091 mEq per liter per pH unit. At 15 mM its capacity is 6.136 mEq per liter per pH unit.

It is worth noting that an acetate buffer in acetic acid pKa (pH 4.76) is equimolar in acetic acid and acetate base. (that is, in pKa, acid and base are present in equal amounts). As a result, the resistance to change in pH (buffer capacity) of an acetate buffer in the pKa of acetic acid is the same for the addition of acid and base. Equilibrium with acid and base is a general characteristic of buffering agents in buffers at a pH equal to their pKa.

At any other pH, a buffer will contain different amounts of acidic and basic forms and therefore its resistance to change (i.e. its buffering capacity) after acid addition will not be the same as its resistance to change after addition. base. As a result, it is preferable to define the capacity of such buffers in terms of (i) the amount of acid necessary to lower the pH by one unit, and (ii) the amount of base necessary to raise the pH by one unit.

The partition in a buffer between the acid and base forms in a given composition, such as a pH standard, can be calculated at any pH and buffer concentration using the procedures set forth above when describing the buffering capacity of 10 mM NaOAc at pH. 4.76 or so (containing equimolar amounts of acetic acid and sodium acetate). And the results can be used to define the buffering capacity of a standard for reference use.

Therefore, for example, The partition of acetic acid in acetic acid and acetate base in a solution at pH 5.0 can be easily calculated using the above procedures, and from this the buffer capacity can be calculated for both the addition both base and acid. Calculated in this way, the theoretical buffering capacity of the 10 mM sodium acetate buffer in the range of pH 5.0 to 5.5 is approximately 2.1 mM per 0.5 pH unit and 4.2 mM per pH unit. pH. In other words, the buffer capacity of the buffer, theoretically, is about 4.2 pEq per ml of buffer per unit of change in pH. Similarly, the theoretical buffering capacity of 10 mM sodium acetate buffer in the range of pH 5.0 to 4.0 is 4.9 mM, and in other words 4.9 pEq per ml of buffer per unit of pH change in a given pH range.

While these calculations are often quite useful in many cases, empirical standards and empirical determinations are preferred. Among the particularly preferred empirical standards are sodium acetate buffers in the range of pH 5.0 to 4.0 and pH 5.0 to 5.5 as exemplified in Examples 1 and 2. Especially preferred are the buffers of sodium acetate according to which the total acetate concentration is, in particular, 10 mM, preferably 5 mM, especially 4 mM, among others as set out elsewhere herein.

Acetate buffers at pH 5.0 are more resistant to pH change by adding acid than by adding base, as discussed above. In a preferred standard buffer capacity, the buffer capacity of a standard acetate buffer such as these is defined as: (i) the slope of the least squares regression line calculated for the baseline titration data for the pH 5 buffer , 0 at pH 5.5, and (ii) the slope of the least squares regression line calculated for the acid titer data for the pH 5.0 pH 4.0 buffer. The preparation of standard acetate buffers and the determination of their buffer capacities are described in Examples 1, 2, and 3. It should be appreciated that the same methods can be used to establish and use buffer capacity standards using other suitable buffering agents.

When measuring the buffering capacity of a self-buffering protein composition according to the invention, it is often convenient to express the buffering capacity in terms of the concentration of a standard buffer at the same pH that has the same buffering capacity. When using a standard that is not in the pKa of the buffering agent, such as a sodium acetate buffer initially at pH 5.0, according to the invention, the self-buffering composition is defined as having equal or greater buffering capacity that of the standard, if its buffering capacity in the base titration or its buffering capacity in the acid titration (or both) is equal to or greater than the corresponding buffering capacity of the standard.

It should further be appreciated that the pH of the self-buffering protein compositions according to the invention will generally not be in the pKa of the self-buffering protein, or of any acid-base substituents therein. In fact, proteins are polyprotic and, as discussed in this paper, will often have several substituents, each with a somewhat different pKa that contributes to its buffering capacity in a given pH range. Accordingly, the buffering capacity of self-buffering protein formulations according to the invention is preferably determined empirically by both acid and base titration over a given range of pH change from the desired pH of the composition. In preferred embodiments in this regard, the buffering capacity is determined by titrating with acid and separately based on a change of respectively ± 1 pH unit from the initial pH of the formulation. In particularly preferred embodiments, the titration data is collected for a change in pH of plus or minus 0.5 pH units. As described in the Examples, the buffer capacity is the slope of the least squares regression line for the pH data as a function of the acid or base equivalents added to the composition over the titration range.

to. Measurements and Empirical Patterns of Buffer Capacity

In certain preferred embodiments of the invention, the buffer capacity measurement is an empirical standard. Among the preferred empirical standards in this regard are a particular volume of an aqueous solution at a particular temperature and pH, containing a particular buffering agent at a particular concentration and having no other components than water, or one or more distinct particular components, each at a particular concentration.

A particularly preferred specific standard for determining buffering capacity according to various aspects and preferred embodiments of the invention is 10 mM sodium acetate, pH 5.00 in pure water with no other constituents at 21 ° C, in equilibrium with ambient air at 1 atmosphere, as described in Examples 1 and 2, preferably expressed in equivalents per unit volume per unit pH, such as pEq / ml-pH. The buffer capacity of the standard should be measured empirically as described in Examples 1, 2, and 3, and as discussed further elsewhere herein.

A particularly preferred specific standard for determining buffering capacity according to various aspects and preferred embodiments of the invention is 10 mM sodium acetate, pH 4.76 in pure water with no other constituents at 21 ° C, in equilibrium with ambient air at 1 atmosphere, as described in Examples 1 and 2, preferably expressed in equivalents per unit volume per unit pH, such as pEq / ml-pH. The buffer capacity of the standard should be measured empirically as described in Examples 1, 2, and 3, and as discussed further elsewhere herein. According to the Henderson-Hasselbalch equation, as indicated above, the calculated buffer capacity of this standard in the range of pH 4.76 plus or minus 1 pH unit is 4.09 microequivalents per milliliter per pH unit (4 , 09 pEq / ml-pH).

A variety of other buffers are available for use as standards in other pH ranges in accordance with various aspects and preferred embodiments of the invention in this regard. Reference buffers are particularly preferred in this regard, such as those well known and commonly used for analytical chemistry determinations. A variety of such buffering agents are set forth in analytical chemistry textbooks and in monographs on the precise determination of pH and buffering capacity.

Also useful in the invention in this regard are biological buffers, such as those described in, inter alia: TEITZ TEXTBOOK OF CLINICAL CHEMISTRY, 3rd ed., Burtis and Ashwood, eds., W.B. Saunders Company, Philadelphia, PA (1999), in particular in Tables 50-13 to 50-16, regarding buffering agents and buffers and their use as pH standards and / or buffer capacity according to the invention in this regard ; THE TO OLS OF BIOCHEMISTRY, Terrance G. Cooper, John Wiley & Sons, New York, NY (1977), in particular Chapter 1, pages 1-35, regarding buffering agents and buffers and their use as pH standards and buffer capacity in accordance with the invention in this regard, more particularly with regard to Tables 1-3, 1-4 and 1-5 and the text related thereto, and PROTEIN PURIFICATION PRINCIPLES AND PRACTICE, 3rd ed., Robert K Scopes, Springer-Verlag, New York, NY (1994), particularly pages 160-164, especially Tables 6.4 and 6.5 and related text, Chapter 12, section 3, pages 324-333, especially in Tables 12-4 and 12-5 and related text, and the entire Appendix C: Buffers for Use in Protein Chemistry, as to buffering agents and buffers and their use in accordance with the invention in this regard.

However, because some gases dissolved in water react with OH 'and / or H3O +, the empirically determined buffer capacity of the standard solution may vary somewhat from the theoretical value. Therefore, the definition of the pattern requires that the solution be in equilibrium with the atmosphere at a pressure of 1 atmosphere. In addition, the buffer standard should be maintained and contacted only with materials that do not alter its components or its buffering capacity, such as those that leach acids, bases, or other reagents that can alter the effective concentration or activity of the acetate buffer of any in a way that alters its buffering capacity. Taking into account the above, the atmospheric equilibrium and the inertia of the container, the buffer capacity of the standard will scale directly and linearly with its volume. Therefore, the 100 ml buffer capacity will be 1/10 of 1.00 liter and the 10 ml buffer capacity will be 1/100 of 1.00 liter. Accordingly, the volume of the standard can be adjusted for comfort of use and then normalized back to 1 liter, as desired.

It may not always be desirable to make the above 10 mM acetate buffer capacity the standard for practical use. However, a variety of other buffer capacity standards can be prepared and used. same way as acetate buffer, using a variety of other buffering agents. Provided the buffer standards are properly prepared, they can be calibrated against the acetate buffer standard described above and then used in practice. Results obtained with such alternative standards can be expressed in terms of the above acetate standard without substantial distortion or error.

The buffer capacity of such alternative standards can also be calibrated by calculation. For this, the buffer capacity of the alternative standard is determined directly and is expressed in mEq per unit volume per unit pH. Determinations based on the alternative standard can be normalized to the acetate standard using the ratio of the buffer capacities expressed in mEq per unit volume per unit pH of the alternative and acetate standards.

Using such methods, which are commonly used in metrology to relate practical standards to a reference standard, the acetate buffer standard described above provides a portable, scalable, reliable and accurate reference for determining the buffering capacity of any composition that can be compared. easily with disparate measurements made on other compositions using similar methods.

b. Preparation of Buffer Capacity Standards

Buffering standards can be prepared using well-established analytical chemistry methods. See, for example, ANALYTICAL c He MISt Ry, 3rd ed., Douglas A. Skoog and Donald M. West, Holt, Rinehart and Winston, New York (1979), particularly chapter 9 (pages 186-226), chapter 10 (pages 227-233) and the methods described on pages 583-588; TEITZ TEXTBo Ok OF CLINICAL CHEMISTRY, 3rd ed., Burtis and Ashwood, eds., W.B. Saunders Company, Philadelphia, PA (1999), particularly Chapter 1 on general laboratory techniques for preparing and calibrating buffers and Tables 50-13 through 50-16; THE TOOLS OF BIOCHEMISTRY, Terrance G. Cooper, John Wiley & Sons, New York, Ny (1977), in particular Chapter 1, pages 1-35, and Tables 1-3, 14, and 1-5 and the text related to them; PROTEIN PURIFICATION PRINCIPLES AND PRACTICE, 3rd ed., Robert K. Scopes, Springer-Verlag, New York, NY (1994), particularly pages 160-164, especially in Tables 6.4 and 6.5 and related text, Chapter 12, section 3, pages 324-333, especially Tables 12-4 and 12-5 and related text, and all of Appendix C: Buffers for Use in Protein Chemistry; and REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 21st Ed., Beringer et al. Editors, Lippincott, Williams & Wilkins, Philadelphia, PA (2005), particularly in the parts related to buffering agents, buffers, buffer capacity and the like, particularly in the preparation and use of buffers and buffer capacity standards according to the invention to this respect.

The water used to prepare the buffer capacity standards should be highly purified, preferably Type I water, such as milliQ water or triple distilled water. Buffer reagents must be pure and, in particular, free of any substance that could alter the pH or buffer capacity of the standard solution, such as reference grade or ACS reagent grade reagents suitable for use in analytical chemical analysis. demanding, as described in the references above, TEITZ and REMINGTON cited above in particular, particularly in parts related to water and analytical grade reagents.

The exact compositions of the buffer reagents must be well established. The molecular weight of the buffer reagents must be precisely known for each buffer reagent. Molecular weights should be for the exact reagent to be used and should include the weight of adducts such as hydrates that are present in the reagent. The effective number of hydrogen donors or hydrogen acceptors per molecule must be precisely known for each buffer reagent. The proportional distribution of different forms, such as hydrates, must be known for each reactant that contains a mixture of such forms. The concentrations of liquid buffer reagents must be known exactly, preferably in moles / volume and in moles / mass (for example, moles / liter and moles / g or kg. Hygroscopic agents should be dried to remove moisture, so that the reagent can be weighed accurately.

Generally speaking, the information provided by well-established vendors of reference grade chemicals and reagents is accurate enough for the preparation of buffer capacity standards as described above. And well-known conventional techniques commonly employed in analytical chemistry can be used to dry "hygroscopic reagents" so that they can be weighed accurately.

As described herein, well-established and commonly used analytical chemistry methods can be used to prepare and calibrate acidic and basic solutions, such as 1N HCl and 1N NaOH (to name just two) to titrate capacity standard solutions. buffer, as well as sample protein solutions, to determine buffer capacity. It should be noted that the preparation of NaOH solutions for titration should be done to eliminate inaccuracies arising from the interaction of certain dissolved gases with basic solutions and the pH-altering effects of their solvation. See, for example, Skoog and West (1979) and other references cited above with respect to the preparation and calibration of buffers and buffer standards, particularly in parts relating to the preparation of standard solutions for titration, as discussed above.

c. Empirical Measures of Buffer Capacity

Titration of standards and samples to determine buffer capacity can be done using standard, well-known methods. Titrations can be carried out manually. They can also be carried out using a self-titrator. A wide variety of autotiters that are suitable for use in the invention in this regard are commercially available from numerous vendors. Methods suitable for use in the invention in this regard are the same as those described in the references cited above with respect to the preparation and calibration of buffer standards, particularly in parts related to the titration of known and unknown solutions to determine their buffer capacity.

2. Protein Buffering and Protein Buffer Capacity

to. Determination of Hydrogen Balance of Proteins and Buffer Capacity

Proteins invariably contain many acidic and basic components. As a result, the balance of hydrogen ions in proteins is highly complex. In fact, a complete description of the hydrogen ion equilibria of a protein in a given environment is beyond the scope of current theoretical and computational methods. Therefore, empirical measurements of protein buffer capacities are preferred. Methods developed for the precise empirical measurement of protein hydrogen balances, which can be routinely employed by those skilled in the art, are suitable for measuring the buffering properties of proteins related to the development of self-buffering protein formulations in accordance with the invention. Therefore, pH titration curves of proteins can be determined in accordance with the invention by well known methods such as those described in and exemplified by the Tanford et al. PH titration studies on ribonuclease. See C. Tanford, "Hydrogen Ion Titration Curves of Proteins," in T. Shedlovsky (ed.), ELECTROCHEMISTRY IN BIOLOGY AND MEDICINE, John Wiley and Sons, New York, 1955, Ch. 13; C. Tanford and J.D. Hauenstein, J. Am. Chem. Soc. 78, 5287 (1956), C. Tanford, PHYSICAL CHEMISTRY OF MACROMOLECUl Es, John Wiley and Sons, New York, 1961, particularly pages 554-567, particularly in parts relating to the titration of proteins with hydrogen ions and with the determination of the buffering action and the buffer capacity of the proteins.

However, the present invention does not need determinations as precise as those described in the above references. Rather, the buffering properties and buffering capacity of the proteins according to the invention can be determined using the methods described in conventional references on analytical chemistry and biochemistry, such as, for example, Skoog (1979), Cooper (1977) and Scopes (1994)., cited above, particularly in regard to the empirical determination of titration curves, particularly of proteins within a given pH range according to the invention.

The determination of titration curves and buffer capacity in accordance with the invention is described in detail for numerous acetate buffers and a variety of pharmaceutical proteins in the Examples below. Therefore, the pH titration curves of the proteins can be empirically determined according to the methods described in the above references on particular limited pH ranges that are of interest for a given formulation. In many respects, these methods are the same as those used in analytical chemistry for the titration of small molecules, such as acetate buffers (as illustrated in the Examples). However, greater care must be taken in handling proteins to maintain the conformation and function necessary for effective formulation.

Protein titrations can be carried out manually or using automated titrators. Equipment for manual titration and automatic titrators are readily available from a large number of vendors and vendors. Suitable methods for determining pH titration curves and protein buffering capacity are exemplified in the Examples by reference to the titration of acetate buffer standards and the titration of several different therapeutic proteins over defined pH ranges. These methods can be used to determine the hydrogen ionization behavior and buffer capacity of any other protein according to the invention.

It is a particular aspect of the invention to determine the buffer capacity of the proteins as a function of the concentration in solution. In a preferred method in this regard, solutions of a given protein are prepared in a graduated series of concentrations. A pH titration curve is determined for the protein at each concentration in the pH range of interest. Preferably, titration curves are determined for the range of interest using both base titration and acid titration. The data, in certain preferred embodiments, are plotted on a graph of added acid or base equivalents versus the measured pH of each solution. Generally, for ranges of about 0.5 to 1.0 pH units, the titration data for each concentration closely fits a straight line, preferably determined by least squares regression analysis. In preferred embodiments in this regard, the buffering capacity for the protein at each concentration will be equated to the slope of the regression line, expressed in equivalent units per ml per pH unit (or fractions thereof). Also useful in the invention in this regard is the relationship between the buffering capacity of the protein and its concentration. In certain preferred embodiments, this relationship is determined by a straight line best fit least squares regression analysis of the buffer capacity data determined in accordance with the above plotted on a plot of buffer capacity versus protein concentration.

The empirical data on the buffering capacity of the proteins according to the invention preferably relates to the buffering capacity of a standard acetate buffer. That is, in particularly preferred embodiments of the invention in this regard, the buffering capacity of a given protein at a given concentration in a given formulation, determined as above, will be equated to the concentration of a standard acetate buffer having the same capacity tampon.

While empirical determinations as described herein are generally a crucial aspect of formulating self-buffering compositions in accordance with various aspects and preferred embodiments of the invention, theoretical and computerized methods can also be productively employed to guide design. , manufacture and use of such compositions (in conjunction with empirical determinations), as described below.

b. Prediction of Hydrogen Ion Balances of Proteins and Buffer Capacity

The ionization of hydrogen in proteins is complex, but can be broken down broadly into pH ranges defined by the ionizable hydrogens of the amino acid side chains and the amino and carboxyl end groups. The pKa of the terminal carboxyl in polypeptides generally ranges around 3.1. The pKa of the acidic hydrogens in the side chains of aspartic acid and glutamic acid ranges around 4.4. The histidine pKa in polypeptides generally ranges around 6.0. The pKa of hydrogen ionization of the terminal amino group generally ranges around 7.5. The sulfhydryl in cysteine has a pKa that ranges around 8.5. Hydroxyl tyrosine and lysine amine have pKa that range around 10. The pKa of arginine ranges around 12.

Conformational folding generally splits large polypeptides and proteins in polar solvents into exposed solvent-accessible regions and into more or less nonpolar core regions that have little or no contact with the environmental environment. Folding produces many environments between these two extremes. Also, the microenvironment around a given amino acid side chain in a protein is generally affected by one or more of: solvent effects; ion bonding; chelation; complex formation; association with cofactors; and post-translational modifications; to name just a few possibilities. Each of these can influence the pKa of a given amino acid ionization in a protein. The pKas for specific residues in a given protein, therefore, can vary dramatically from that of a free amino acid.

Indeed, alteration of pKa by amino acid microenvironments in proteins has been used to study protein folding and the arrangement and charge state of specific amino acids in folded proteins. The protein titer curves reported by Tanford and others are complex with some general features in common. Generally, only some of the ionizable protons are accounted for in the titration curves. Others are apparently located in the core and are inaccessible to the solvent. Individual side chain pKas of the same type that can be detected in some cases can be distinguished from each other. However, while they are detectably different, their pKas are generally similar to those of the free amino acid.

The strongest buffering action of proteins generally does not occur at the isoelectric point, as may be erroneously assumed. In fact, buffering is dependent on amino acid side chain hydrogens and terminal hydrogens, and therefore occurs in ranges spanning the pKas of ionizable hydrogens in free amino acids, as discussed above. The most important of these, for formulating protein compositions, especially certain pharmaceutical proteins that are more soluble and / or more stable, among other things, at weakly acidic pH (pH 4 to 6), is the buffering action that occurs in the range of the carboxylic hydrogen pKa of the amino acids aspartic acid and glutamic acid; that is, pH 4.0 to 5.5, particularly around 4.5.

There are a variety of ways available to estimate the buffering capacity of a given protein in a given solution at a given pH. The methods range from highly technical and complex computer models to those that can be carried out on a manual calculator. Neither method is complete or totally accurate; but, in some cases, they can provide useful estimates.

For example, in some cases, a potentially useful idea of the buffering capacity for a protein in a solution can be calculated based on its amino acid composition, the pKa (in the solvent in question) of terminal amino and carboxy groups, and donors and hydrogen acceptors of amino acid side chains, the concentration of the protein and the pH of the solution.

For example, a potentially useful estimate of the buffering capacity of a protein at pH in the range of the pKa of the carboxylic hydrogen side chain of glutamic acid (as free amino acid) can be obtained from the molecular weight of the protein and the number of glutamic acid residues it contains. Divide the first by the last provides the weight per equivalent of glutamic acid and thus the weight per equivalent of ionizable hydrogen in the pKa of glutamic acid. Because the side chain carboxyl groups of glutamic acid and aspartic acid have almost the same pKa, the results of such calculations for the two must be added together to obtain an estimate of the buffering capacity in an interval around their two pKa. The estimated buffer capacity of a solution of the protein in pKa can be calculated from the concentration of protein in the solution and the intrinsic factor just provided, that is, the weight per equivalent of ionizable hydrogen. Dividing the concentration by weight per equivalent gives an estimate of the buffer capacity in units of Eq / volume. Such estimates will often be too high, since some residues are generally sequestered in regions of the protein that are not accessible to the solvent and therefore do not contribute to its true buffering capacity. It may be possible in certain cases to take into account the effect of sequestration on the buffer capacity. For example, a fractional coefficient that reflects theoretical or empirical estimates of sequestration can be applied to fit the original calculation.

Such calculations will generally be less useful and less accurate than empirical determinations of the buffering capacity of proteins, according to the methods described herein. But they can be useful in providing approximate maximum estimates of the buffering capacity of proteins in solution.

3. Proteins

The present disclosure can be practiced with any protein that provides sufficient buffering capacity in a desired pH range within the parameters of protein concentration and the like necessary for a desired formulation. Among the preferred proteins in this regard are pharmaceutical proteins for veterinary and / or human therapeutic use, particularly proteins for human therapeutic use. Also among the preferred proteins are proteins that are soluble in aqueous solutions, particularly those that are soluble in relatively high concentrations and those that are stable for long periods of time. Additionally, preferred proteins include those having a relatively high number of solvent accessible amino acids with side chain hydrogen ionization constants near the pH of the desired buffering action.

Furthermore, among the preferred proteins of the disclosure are proteins for pharmaceutical formulations that do not induce a highly detrimental antigenic response after administration to a subject. In this regard, proteins for veterinary and / or human medical use are preferred, particularly, relative to the latter, humanized and human proteins.

Furthermore, among the preferred proteins of the disclosure are proteins that selectively bind to specific targets, including ligand-binding proteins and protein ligands. Antigen-binding proteins, proteins derived therefrom, and proteins related thereto are among the particularly preferred embodiments of the disclosure in this regard. Highly preferred proteins of the disclosure in this regard are antibodies and antibody-derived or antibody-incorporating proteins, in whole or in part, including, to name just a few of those entities: monoclonal antibodies, polyclonal antibodies, engineered antibodies. genetics, hybrid antibodies, bispecific antibodies, single chain antibodies, genetically altered antibodies, including antibodies with one or more amino acid substitutions, additions and / or deletions (antibody muteins), chimeric antibodies, antibody derivatives, antibody fragments, which can be of any of the above and may also be similarly engineered or modified derivatives thereof, fusion proteins comprising an antibody or a moiety derived from an antibody or from an antibody fragment, which may be any of the above or a modification or derivative of the m ism, conjugates comprising an antibody or an antibody-derived moiety, including any of the foregoing, or modifications or derivatives thereof, and chemically modified antibodies, antibody fragments, antibody fusion proteins, and the like, including all of the foregoing .

to. Antibodies, Antibody-Derived and Antibody-Related Proteins and the Like

Among the particularly preferred proteins according to the disclosure are antibody polypeptides, such as heavy and light chain polypeptides that have the same amino acid sequence as those that occur and form naturally occurring antibodies, such as those that are produced in sera and antisera, including polypeptides and proteins isolated from natural sources, as well as those made by hybridoma technologies, by activating an endogenous gene (by homologous or non-homologous recombination, for example), by expressing of an exogenous gene under the control of an endogenous transcriptional control region, by expression of an exogenous expression construct, by semisynthesis and by de novo synthesis , to name a few techniques commonly used to prepare antibodies and related polypeptides and proteins. antibodies that can be used to make polypeptides and proteins of antibodies according to the disclosure.

These antibody-related polypeptides and proteins include those that, in whole or in part, have a de novo amino acid sequence , those that comprise all or one or more parts of a antibody (that is: a continuous chain of amino acids that has the same sequence as any four or more residues in the amino acid sequence of a naturally-occurring antibody polypeptide), those that have an amino acid sequence that somehow matches the of a naturally occurring antibody, but differs from it in other ways, those that have the same but different amino acid sequences to a homolog or naturally occurring sequence that is related to them, but differ from the homologue in one or more post-translational modifications, and those comprised in part of any of the foregoing (in part or in whole) fused to one or more polypeptide regions that may be from or derived from or related to a second different antibody polypeptide, and may be or derived from any other polypeptide or protein, whether of natural origin, similar but differing from them, having a semi-amino acid sequence -de novo and / or a de novo sequence , among others. Such hybrids are generally referred to herein as fusion polypeptides and / or fusion proteins.

Furthermore, among the preferred proteins in accordance with the disclosure described herein, are proteins modified in accordance with all of the above. Included among such modified proteins are proteins chemically modified by a non-covalent bond, covalent bond, or both a covalent bond and a non-covalent bond. Also included is all of the foregoing that further comprises one or more post-translational modifications that can be performed by cellular modification systems or modifications introduced ex vivo by enzymatic and / or chemical methods, or introduced in other ways.

Among the preferred proteins of the disclosure in this regard are Fab fragments, such as those produced by cleaving a typical dimeric (LH) 2 antibody with a certain protease that leaves the light chain intact while the heavy chains between the variable region and adjacent constant region, "on" the disulfide bonds that hold the heavy chains together. Such cleavage releases one Fc fragment comprising the remaining portions of the heavy chains linked together, and two dimeric Fab fragments each comprising an intact light chain and the variable region of the heavy chain. Fab fragments can also be produced by other techniques that do not require isolation of a naturally occurring antibody and / or cleavage with a protease.

Also preferred are Fab2 fragments such as those produced in much the same way as Fab fragments using a protease that cleaves "between or below" disulfide bonds. As a result, the two Fab fragments are held together by disulfide bonds and released as a single Fab2 fragment. Fab2 fragments can be produced by many different techniques, including those that do not require isolation of an intact antibody or cleavage with a protease that has the necessary specificity. Also, both mono and bispecific Fab2 fragments can now be prepared by a variety of common techniques.

Also among preferred proteins in this regard are Fab3 fragments, which are genetically engineered antibody fragments in which three Fab fragments bind to each other. Fab3 fragments can be mono, bi-, or tri-specific. They can be prepared in a variety of ways well known to those skilled in the related art. Among other preferred proteins in this regard are Fc fragments, such as those produced by cleavage with a protease in the same manner used for the production of either Fab fragments or Fab2 fragments. However, for the production of Fc fragments, fragments containing dimeric heavy chains are isolated rather than fragments containing light chains. Fc fragments lack antigen combining sites, but comprise effector regions that play a role in physiological processes involving antibodies. Fc fragments can be prepared by a variety of techniques that are well known and commonly employed by those skilled in the art for this purpose.

Among other preferred proteins in this regard are single chain variable fragments ("scFv"). ScFvs are fusion proteins prepared by joining the variable regions of the heavy and light chains of an immunoglobulin. The heavy and light chains in an scFv are generally linked by a short serine and glycine linker. The scFv have the same specificity as the antibodies from which they were derived. Originally produced via phage display, scFv can now be made by a variety of well-known methods.

Bis-scFvs which are fusions of two scFvs are also preferred. Bis-scFv can be mono- or bi-specific. A variety of methods are well known and can be applied to prepare Bis-scFv in accordance with the disclosure.

Also preferred according to the disclosure in this regard are minibodies; mono and bispecific diabodies; mono-, bi-, and tri-specific tri-bodies; mono, bi-, tri-, and tetra-specific tetrabodies; VhH domains; V-NAR domains; V ^h domains; V ^l domains; Camel Ig; Ig NAR; and others.

Also among the preferred embodiments according to various aspects and preferred embodiments of the disclosure in these and other aspects are proteins comprising one or more CDR and / or CDR-derived and / or CDR-related regions of an antibody or one or more FR and / or FR-derived and / or FR-related regions of an antibody. In this regard, CDR stands for complementarity determining region; that is, a hypervariable region of an antibody heavy or light chain, generally about 9 to 12 amino acids in length, which is normally a major part of an antigen-specific binding moiety of an antibody. FR in this regard means a framework region of an antibody; that is, a region of approximately 15 to 20 amino acids that separate CDRs into the antigen-specific binding moiety of an antibody. The expressions derived from CDR and related to CDR, and the expressions derived from FR and related to FR have the same meanings for CDR and FR, respectively, as set out in the Glossary above for the expressions derived from antibodies and related to antibodies as al term antibody.

With respect to antibodies, antibody-derived and antibody-related proteins in accordance with the foregoing and other aspects of the disclosure, see, for example, Protein Engineering: Principles and Practice, Jeffrey L. Cleland and Chares S. Craik, eds . Wiley-Liss, Inc., New York (1996), particularly in Kelley, Robert F., "Engineering Therapeutic Antibodies," Chapter 15, pp. 399-434 and Hollinger, P. & Hudson, P., "Engineered antibody fragments and the rise of single domains," Nature Biotechnology, September 2005, 1126-1136, particularly in parts related to the structure and engineering of antibodies, particularly biopharmaceutical antibodies, and antibody-derived and antibody-related proteins, particularly antibody-derived and antibody-related pharmaceutical proteins according to the invention described herein.

Regarding all of the foregoing, particularly preferred in the disclosure are human, humanized and other proteins that do not elicit significantly detrimental immune responses when administered to a human. Also preferred in the disclosure are proteins according to all of the foregoing that similarly do not cause significantly deleterious immune responses on administration to non-humans.

Among the very particularly preferred proteins according to the disclosure in this regard are fusion proteins comprising antibodies and / or antibody-derived proteins, polypeptides or fragments or the like, including all those described above. Among the very particularly preferred fusion proteins of the disclosure in this regard are fusion proteins comprising an antibody or an antibody-derived protein or fragment as described above and a ligand-binding moiety, as illustratively described. in the present document.

b. Target Binding Proteins

Also among the preferred proteins of the disclosure in this regard are antibodies and other types of target-binding proteins, and proteins related thereto or derivatives thereof, and protein ligands, and proteins derived from or related to the same. Among the especially preferred ligand-binding proteins in this regard are proteins that bind signal and effector proteins, and proteins related to or derived therefrom.

Among such binding proteins, including antibodies, including proteins derived therefrom and proteins related thereto, are those that bind to one or more of the following, alone or in any combination:

(i) CD proteins including but not limited to CD3, CD4, CD8, CD19, CD20, and CD34;

ii) proteins of the HER receptor family, including, for example, HER2, h ER3, HER4 and the EGF receptor; (iii) cell adhesion molecules, eg, LFA-1, Mol, p150.95, VLA-4, ICAM-1, VCAM, and alpha v / beta 3 integrin;

(iv) growth factors, including but not limited to, for example, vascular endothelial growth factor ("VEGF"); growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, Mullerian inhibitory substance, human macrophage inflammatory protein (MIP-1-alpha), erythropoietin (EPO), factor of nerve growth, such as NGF-beta, platelet-derived growth factor (PDGF), fibroblast growth factors, including, for example, aFGF and bFGF, epidermal growth factor (EGF), transforming growth factors (TGF), including, but not limited to, TGF-alpha and TGF-beta, including TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4 or TGF-beta5, insulin-like growth factors I and II (IGF-I and IGF-II ), des (1-3) -IGF-I (brain IGF-I) and osteoinductive factors;

(v) insulins and insulin-related proteins, including but not limited to insulin, insulin A chain, insulin B chain, proinsulin, and insulin-like growth factor binding proteins;

(vi) coagulation and coagulation-related proteins, such as, but not limited to, factor VIII, tissue factor, von Willebrand factor, protein C, alpha-1 antitrypsin, plasminogen activators, such as urokinase, and tissue plasminogen activator ( "t-PA"), bombazine, thrombin and thrombopoietin;

(vii) colony stimulating factors (CSF), including the following, but not limited to, M-CSF, GM-CSF, and G-CSF;

(viii) other blood and serum proteins, including but not limited to albumin, IgE, and blood group antigens;

(ix) receptors and receptor-associated proteins, including, for example, flk2 / flt3 receptor, obesity receptor (OB), growth hormone receptors, and T-cell receptors;

(x) neurotrophic factors, including but not limited to, bone derived neurotrophic factor (BDNF) and neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6 );

(xi) relaxin A chain, relaxin B chain, and prorelaxin;

(xii) interferons, including, for example, interferon-alpha, -beta, and -gamma;

(xiii) interleukins (IL), eg, IL-1 to IL-10;

(xiv) viral antigens, including but not limited to a viral AIDS envelope antigen;

(xv) lipoproteins, calcitonin, glucagon, atrial natriuretic factor, lung surfactant, tumor necrosis factor alpha and beta, enkephalinase, RANTES (normally regulated in the activation of expressed and secreted T lymphocytes), mouse gonadotropin-associated peptide, DNase, inhibin and activin;

(xvi) Integrin, protein A or D, rheumatoid factors, immunotoxins, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane proteins, decomposition accelerating factor (DAF), AIDS envelope, carrier proteins, targeted migration receptors , adresins, regulatory proteins, immunoadhesins, antibodies; and

(xvii) Biologically active fragments or variants of any of the above.

Regarding all of the foregoing, particularly preferred are those that are effective therapeutic agents, particularly those that exert a therapeutic effect by binding to a target, particularly a target among those listed above, including targets derived therefrom, related targets with the same and modifications thereof.

C. Particular Illustrative Proteins

Particular illustrative proteins include certain antibodies and antibody-related proteins, including peptibodies, such as, for example, those listed immediately below and elsewhere herein:

OPGL-specific antibodies and peptibodies and the like (also referred to as antibodies, RANKL-specific peptibodies and the like, including fully humanized and human OPGL-specific antibodies, particularly fully humanized monoclonal antibodies. In particular, the invention includes the antibodies described in International Publication number WO 03/002713, which is incorporated herein, as OPGL-specific antibody-related antibodies and proteins, particularly those having the sequences set forth therein, particularly, but not limited to, those set forth therein: 9H7; 18B2; 2D8; 2E11; 16E1; and 22B3, including OPGL-specific antibodies having the heavy chain of SEQ ID NO: 2 as set forth in Figure 2 (thereby producing the heavy chain of the antibody) and / or the light chain of SEQ ID NO: 4 (thereby producing the antibody light chain), as stated in Figure 4, each of which is individually and specifically incorporated by reference herein in its entirety, as disclosed in the foregoing publication. Acid and base titers of an OPGL specific antibody ("Ac-hOPGL") in the pH ranges 4.5 to 5.0 and 5.0 to 5.5 are described in the Examples below. The calculation of the Ac-hOPGL buffer capacity in these pH ranges is also described in the Examples below.

Myostatin-binding agents or peptibodies, including myostatin-specific peptibodies, particularly those described in U.S. Application Publication No. 2004/0181033, particularly in parts related to myostatin-specific peptides, including, but not limited to, without limitation, peptides of the mTN8-19 family, including those of SEQ ID NO: 305-351, including TN8-19-1 through t N8-19-40, TN8-19 conl and TN8-19 con2; peptides of the ml2 family of SEQ ID NO: 357-383; the ml15 family of SEQ ID NO: 384409; the ml17 family of SEQ ID NO: 410-438; the ml20 family of SEQ ID NO: 439-446; the ml21 family of SEQ ID NO: 447-452; the ml24 family of SEQ ID NO: 453-454; and those of SEQ ID NO: 615-631.

Specific IL-4 receptor antibodies, particularly those that inhibit activities mediated by IL-4 and / or IL-13 binding to the receptor, including those described in International Publication No. WO 2005/047331 of International Application Number PCT / US2004 / 03742, particularly in parts related to the IL-4 receptor specific antibodies, particularly the antibodies described therein, particularly, and without limitation, those designated therein: L1H1; L1H2; L1H3; L1H4; L1H5; L1H6; L1H7; L1H8; L1H9; L1H10; L1H11; L2H1; L2H2; L2H3; L2H4; L2H5; L2H6; L2H7; L2H8; L2H9; L2H10; L2H11; L2H12; L213; L2H14; L3H1; L4H1; L5H1; L6H1. Acid and base titrations over the pH ranges 4.5 to 5.0 and 5.0 to 5.5, and the calculation of the buffering capacity in this range of an antibody specific for the IL-4 receptor (" Ac-hIL4R ") are described in the examples below.

Interleukin 1 receptor 1 ("IL1-R1") specific antibodies, peptibodies and related proteins, and the like, including, but not limited to, those described in United States Application Publication Number US2004 / 097712A1, in related parts IL1-R1 specific binding proteins, monoclonal antibodies in particular, especially, without limitation, those designated therein: 15CA, 26F5, 27F2, 24E12 and 10H7.

Antibodies and peptibodies specific to Ang2 and related proteins and the like, including but not limited to those described in International Publication Number WO 03/057134 and in United States Application Publication Number US2003 / 0229023, particularly in parts related to antibodies and peptibodies specific to Ang2 and the like, especially those of sequences described therein and including but not limited to: L1 (N); L1 (N) TS; L1 (N) 1K TS; 2xL1 (N); 2xL1 (N) TS; Con4 (N), Con4 (N) 1K TS, 2xCon4 (N) 1K; L1 (C); L1 (C) 1K; 2xL1 (C); Con4 (C); Con4 (C) 1K; 2xCon4 (C) 1K; Con4-L1 (N); Con4-L1 (C); TN-12-9 (N); C17 (N); TN8-8 (N); TN8 14 (N); With 1 (N), including also anti-Ang 2 antibodies and formulations such as those described in International Publication Number WO 2003/030833, particularly Ab526; Ab528; Ab531; Ab533; Ab535; Ab536; Ab537; Ab540; Ab543; Ab544; Ab545; Ab546; A551; Ab553; Ab555; Ab558; Ab559; Ab565; AbF1AbFD; AbFE; AbFJ; AbFK; AbG1D4; AbGC1E8; AbH1C12; AblAl; AblF; Ab1KAb1P; and blP, in its various permutations as described therein.

NGF-specific antibodies, including, in particular, but not limited to, those described in United States Application Publication Number US2005 / 0074821, particularly regarding antibodies specific to NGF and related proteins in this regard, including in particular, but without limitation, the NGF-specific antibodies therein designated 4D4, 4G6, 6H9, 7H2,14D10 and 14D11.

CD22-specific antibodies and related proteins, such as those described in US 5,789,554 for CD22-specific antibodies and related proteins, particularly human CD22-specific antibodies, such as, but not limited to, humanized and fully human antibodies, including, but not limited to, humanized and fully human monoclonal antibodies, including particularly, but not limited to, human CD22-specific IgG antibodies, such as, for example, a human-mouse monoclonal hLL2 gamma chain disulfide dimer bound to a human-mouse monoclonal kappa chain hLL2, including, but not limited to, for example, the fully humanized human CD22-specific antibody to Epratuzumab, CAS registry number 501423-23-0. Illustrative of the invention, the acid and base titrations of a CD22-specific antibody ("Ac-hCD22") in the pH ranges 4.5 to 5.0 and 5.0 to 5.5 are described in Examples a continuation. The calculation of the Ac-hCD22 buffering capacity in these pH ranges is also described in the Examples below.

Specific antibodies to the IGF-1 receptor and related proteins such as those described in International Patent Application Number PCT / US2005 / 046493, regarding specific antibodies to the IGF-1 receptor and related proteins, including, but not limited to, the IGF-1 specific antibodies therein designated L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13, L14H14, L15H15, L16H16, L17H20, L20H19 , L22H22, L23H23, L24H24, L25H25, L26H26, L27H27, L28H28, L29H29, L30H30, L31H31, L32H32, L33H33, L34H34, L35H35, L36H36, L37H37, L38H38, L39H39, L40H43, L42H44, L42H43, L42H43, L42H43, L42H43 , L47H47, L48H48, L49H49, L50H50, L51H51 and L52H52.

Specific antibodies to B-7 related protein 1 ("B7RP-1"), (B7RP-1 is also known in the literature as B7H2, ICOSL, B7 h and CD275), particularly B7RP-specific monoclonal fully human IgG2 antibodies, particularly fully human monoclonal IgG2 antibody that binds to an epitope in the first immunoglobulin-like domain of B7RP-1, especially those that inhibit the interaction of B7RP-1 with its natural receptor, ICOS, in activated T lymphocytes in particular, especially, in all of the foregoing aspects, those disclosed in United States Provisional Application Number 60 / 700,265, filed July 18, 2005 as such antibodies and related proteins, including but not limited to the antibodies designated therein as follows: 16H (which it has the variable light chain and variable heavy chain sequences, SEQ ID NO: 1 and SEQ ID NO: 7 respectively in it); 5D (having the variable light chain and variable heavy chain sequences, SEQ ID NO: 2 and SEQ ID NO: 9 respectively therein); 2H (having the variable light chain and variable heavy chain sequences, SEQ ID NO: 3 and SEQ ID NO: 10 respectively therein); 43H (having the variable light chain and variable heavy chain sequences, SEQ ID NO: 6 and SEQ ID NO: 14 respectively therein); 41H (having the variable light chain and variable heavy chain sequences, SEQ ID NO: 5 and SEQ ID NO: 13 respectively therein); and 15H (having the variable light chain and variable heavy chain sequences, SEQ ID NO: 4 and SEQ ID NO: 12 respectively therein). Acid and base titrations and determination of the buffering capacity of a B7RP-1 specific antibody ("Ac-hB7RP1") are illustrated in the Examples below.

IL-15-specific antibodies, peptibodies and related proteins, such as, in particular, humanized monoclonal antibodies, particularly antibodies as disclosed in US Application Publication Numbers: US2003 / 0138421; US2003 / 023586; US2004 / 0071702 for antibodies specific to IL-15 and related proteins, including peptibodies, including particularly, for example, but not limited to, IL-15 HuMax antibodies and related proteins, such as, for example, 146B7.

Specific antibodies to IFN gamma, especially human IFN gamma specific antibodies, particularly anti-fully human IFN gamma antibodies, such as, for example, those described in US Application Publication Number US2005 / 0004353 regarding specific antibodies from IFN gamma, particularly, for example, the antibodies therein designated 1118; 1118 *; 1119; 1121; and 1121 *.

TALL-1 specific antibodies and other TALL-specific binding proteins such as those described in United States Application Publication Number 2003/0195156, regarding TALL-1 binding proteins, particularly the molecules in Tables 4 and 5B.

Stem cell factor (s) ("SCF") and related proteins such as those described in United States Patent Nos. 6,204,363 and 6,207,802, for stem cell factors and related proteins , particularly, for example, the stem cell factor "STEMGEN ™".

Flt3 ligands, ("Flt3L") and related proteins such as those described in US Patent Number 6,632,424, as to Flt3 ligands and related proteins in this regard.

IL-17 Receptors and Related Proteins ("IL-17R"), such as those described in US Patent Number 6,072,033, for Flt3 ligands and related proteins in this regard.

Etanercept, also known as Embrel and related proteins.

Actimmune (Interferon-gamma-lb), Activase (Alteplase), Aldurazme (Laronidase), Amevive (Alefacept), Avonex (Interferon beta-1a), BeneFIX (Nonacog alfa), Beromun (Tasonermina), Beatseron (Interferon-beta-lb ), BEXXAR (Tositumomab), Tev-Tropin (Somatropin), Bioclate or RECOMBINATE (Recombinant), CEREZME (Imiglucerase), ENBREL (Etanercept), Eprex (epoetin alfa), EPOGEN / Procit (Epoetin alfa), FAb Raalsida ZYME (FAb Raalsida ZYME beta), Fasturtec / Elitek ELITEK (Rasburicase), f Or TEO (Teriparatide), GENOTROPIN (Somatropin), GlucaGen (Glucagon), Glucagon (Glucagon, rDNA origin), GOnAl-F (follitropin alfa), KOGENATE FS (Octocog alfa ), HERCEPTIN (Trastuzumab), HUMATROPE (SOMATROPIN), HUMIRA (Adalimumab), Insulin Solution, INFERGEN® (Interferon alfacon-1), KINEr Et ® (anakinra), Kogenate Fs (Antihemophilic Factor), LEUKIN (SARGRAMOSTIM stimulating factor Recombinant Human Granulocyte-Macrophage Colonies (rhuGM-CSF)), CAMPATH (Alemtuzumab), RITUXAN® (Rituximab), TNKase (Tenecteplase), MY LOTARG (gemtuzumab ozogamicin), NATRECOR (nesiritide), ArA n ESP (darbepoetin alfa), NEULASTA (pegfilgrastim), NEUMEGA (oprelvekin), NEUPOGEN (Filgrastim), NORDITROPIN CARTRIDGES (SomaVENtropin, AUcoVOSEtOPIN), NOcoVOSEtOPIN (somatropin), Oncaspar (pegaspargasse), ONTAK (denileucine diftitox), ORTHOCLONE OKT (muromonab-CD3), OVIDREL (choriogonadotropin alfa), PEGAs Ys (peginterferon alfa-2a), PROLEUKIN (Aldesleukasa alfa), PULMOaseZME (dULMOaseZasa) (Reteplase), REBETRON Combination Therapy containing REBETOL® (Ribavirin) and INTRON® A (Interferon alfa-2b), REBIF (interferon beta-1a), REFACTO (Antihemophilic Factor), REFLUDAN (lepirudin), REMIc Ad E (infliximab ), REOPRO (abciximab) ROFERON®-A (Interferon alfa-2a), SIMULECT (baasiliximab), SOMAVERT (Pegivisomant), SYNAGIS® (palivizumab), Stemben (Ancestima, Stem cell factor), THYROGEN, INTRON® A (Interfer alfa-2b), Pe G-INTRON® (Peginterferon alfa-2b), XIGRIS® (Drotrecogin alfa activated), XOLAIR® (Omalizumab ), ZENAPa X® (daclizumab), ZEVALIN® (Ibritumomab Tiuxetan).

d. Sequence variation

Particularly preferred proteins with respect to all of the above disclosure and the following disclosure include those that comprise a region that is 70% or more, especially 80% or more, more especially 90% or more, even more especially 95% or more, particularly 97% or more, more particularly 98% or more, even more particularly 99% or more identical in amino acid sequence to a reference amino acid sequence of a binding protein, such as illustrated above, particularly a pharmaceutical binding protein, such as a GenBank or other reference sequence of a reference protein.

Identity in this regard can be determined using a variety of well known and readily available amino acid sequence analysis software. The preferred software includes those that implement the Smith-Waterman algorithms, considered a satisfactory solution to the problem of sequence search and alignment. Other algorithms can also be employed, particularly when speed is an important consideration. Commonly employed programs for DNA, RNA, and polypeptide homology alignment and matching that can be used in this regard include FASTA, TFASTA, BLASTN, BLASTP, BLASTX, TBLAs Tn, p Ro SRc H, BLa Ze, and MPSRCH, being , the latter, an implementation of the Smith-Waterman algorithm for execution on massively parallel processors made by MasPar.

The BLASTN, BLASTX, and BLASTP programs are among the preferred programs for such determinations, the former for polynucleotide sequence comparisons and the latter two for polypeptide sequence comparisons: BLASTX for comparison of the polypeptide sequences of the three frames of read the polynucleotide sequence and BLASTP for a single polypeptide sequence.

BLAST provides a variety of user-definable parameters that are set before implementing a comparison. Some of them are more apparent than others in graphical user interfaces, such as those provided by NCBI BLAST and other sequence alignment programs that can be accessed on the Internet. The settings and their values are set out and explained on the service websites and are explained and set forth in particular detail in a variety of readily available texts, including but not limited to BIOINFORMATICS: SEQUENCE AND GENOME ANALYSIS, 2nd Ed., David W. Mount, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2004), especially Chapters 3, 4, 5 and 6 regarding the comparison of protein and nucleic acid sequences in general and regarding the comparisons and searches BLAST in particular; SEQUENCE ANALYSIS IN A NUTSHELL: A GUIDE TO COMMON TOOLS AND DATABASES, Scott Markel and Darryl Leon, O'Reilly & Associates, Sebastopol, California (2003), especially Chapter 7 regarding BLAST in particular, particularly in the parts related to the comparison of nucleotide and polypeptide sequences and to determine their degree of identity, similarity, homology and / or the like, especially in terms of comparison of a test sequence and a reference sequence to calculate a degree (percentage) of identity between them.

In preferred embodiments of the invention in this regard, sequence ratio is defined as the percent identity score returned by one or other of the aforementioned BLAST comparison searches with e = 10 and all other parameters set to their values. defaults on the NCBI web server as set forth in SEQUENCE ANALYSIS IN A NUTSHELL: A GUIDE TO COMMON TOOLS AND DATABASES, Scott Markel and Darryl Leon, O'Reilly & Associates, Sebastopol, California (2003), pages 47-51 and in full details of the preferred settings for the parameters of the present invention to compare sequences using BLAST, such as those of NCBI BLAST.

The following references provide additional information on sequence comparisons in this regard and elsewhere. GUIDE TO HUMAN GENOME COMPUTING, Ed. Martin J. Bishop, Academic Press, Harcourt Brace & Company Publishers, New York (1994), particularly in the parts related to the determination of the identity and / or homology of amino acid or polynucleotide sequences, especially Chapter 7. BLAST programs are described in Altschul et al., "Basic Local Alignment Research Tool," J Mol Biol 215: 403-410 (1990). Additional information on sequence analysis and homology and identity determinations is provided in, among many other references well known and readily available to those of skill in the art: NUCLEIC ACID AND PROTEIN SEQUENCE ANALYSIS: A PRACTICAL APPROACH, Eds. M. J. Bishop and C. J. Rawings, IRL Press, Oxford, UK (1987); PROTEIN STRUCTURE: A PRACTICAL APPROACH, Ed. T. E. Creighton, IRL Press, Oxford, UK (1989); Doolittle, R. F .: "Searching through sequence databases," Met Enz. 183: 99-110 (1990); Meyers and Miller: "Optimal alignments in linear space" Comput. Applica. in Biosci 4: 11-17 (1988); Needleman and Wunsch: "A general method applicable to the search for similarities in amino acid sequence of two proteins," J Mol Biol 48: 443-453 (1970) and Smith and Waterman "Identification of common molecular subsequences," J Mol Biol 147 : 1950 et seq. (1981), particularly in parts related to the comparison of sequences and determinations of identity and homology.

Particularly preferred embodiments in this regard have 50% to 150% of the activity of the reference protein mentioned above, particularly highly preferred embodiments in this regard have 60% to 125% of the activity of the reference protein, the Even more highly preferred embodiments have 75% to 110% of the activity of the reference protein, still more highly preferred embodiments have 85% to 125% of the activity of the reference, still more highly preferred embodiments have 90 % to 110% of the activity of the reference.

4. Formulations

Many reagents and methods conventionally employed for the formulation of protein pharmaceuticals can be used for the formulation of self-buffering protein compositions in accordance with various aspects and preferred embodiments of the disclosure. However, in self-buffering protein formulations according to the disclosure, the buffering is provided substantially entirely by the protein itself, not by a buffering agent, as is the case with conventional formulations. Furthermore, self-buffering protein formulations according to various aspects and preferred embodiments of the disclosure are substantially free of such buffering agents.

However, in many other respects, self-buffering protein compositions according to various aspects and embodiments of the disclosure can be formulated using reagents and methods conventionally employed for the formulation of proteins, in particular, reagents and methods employed for the formulation of pharmaceuticals. , including pharmaceuticals for veterinary and human use, especially those reagents and methods suitable for formulating protein pharmaceuticals for veterinary use and especially for human use.

Accordingly, many methods and ingredients can be used to formulate and use pharmaceuticals that are well known and common in the related arts, in the design, preparation and use of self-buffering protein formulations in accordance with various aspects and preferred embodiments of disclosure in connection with this. Such methods and ingredients are described in, to name just a few readily available references in this regard, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 21st Ed .; Beringer et al. Editors, Lippincott, Williams & Wilkins, Philadelphia, PA (2005); ANSEL'S PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 8th Edition, Allen et al., Editors, Lippincott, Williams & Wilkins, Philadelphia, PA (2005); and PHARMACEUTICAL FORMULATION OF PEPTIDES AND PROTEINS, Sven Frokjaer and Lars Hovgaard, Editors, CRC Press, Boca Raton, Florida (2000), particularly in the parts related to ingredients and conventional methods that can be used in protein formulations of autobuffers in accordance with various aspects and preferred embodiments of the invention related thereto.

Additional methods and ingredients that may be useful in this regard are described in, among others, US 6,171,586; WO 2005/044854; US 6,288,030; US 6,267,958; WO 2004/055164; US 4,597,966; US 2003/0138417; the US document 6,252,055; US 5,608,038; US 6,875,432; US 2004/0197324; WO 02/096457; US 5,945,098; US 5,237,054; US 6,485,932; US 6,821,515; US 5,792,838; US 5,654,403; US 5,908,826; EP 0804 163; and WO 2005/063291, particularly in the parts related to pharmaceutically acceptable self-buffering protein formulations according to the invention.

Various specific aspects of ingredients and specific types of formulations are further described below, by way of illustration. The description thus provided is not exhaustive of the possible methods and compositions for self-buffering protein formulations in accordance with the various aspects and embodiments of the disclosure, nor is it in any way exclusive.

In preferred embodiments of a variety of aspects of the disclosure, self-buffering protein formulations comprise a protein and a carrier, which may also be variously referred to herein, as the case may be, as one or more of: a carrier , a primary carrier, a diluent, a primary diluent, a primary carrier, a solvent and / or a primary solvent. In the broadest sense, the carrier can be a gas, a liquid or a solid, as appropriate to the phase of the composition and / or its use (s). In some embodiments of the disclosure in this regard, the carrier is a solid, such as a powder, in which a protein can be dispersed. In preferred embodiments in this regard, the carrier is a liquid, particularly a liquid in which the self-buffering protein is highly soluble, particularly at concentrations that provide the desired buffering capacity. Liquid carriers can be organic or non-organic. They are preferably aqueous, most preferably they are composed largely or entirely of pure water.

It will be appreciated that formulations for pharmaceutical use in accordance with various aspects and embodiments of the invention must be compatible with the processes and conditions to which they will be subjected, such as, for example, sterilization procedures (generally applied prior to mixing with a principle active), and conditions during storage.

Almost invariably, formulations according to numerous aspects and embodiments of the invention will contain additional ingredients including, but not limited to, excipients and other pharmaceutical agents. However, it is to be understood that the formulations according to the invention are self-buffering formulations in which the buffering capacity is provided substantially or completely by the primary protein itself, as described elsewhere herein.

Formulations according to various aspects and embodiments of the invention may contain, but are not limited to, excipients, as described below, including but not limited to ingredients to modify, maintain, or preserve, for example, osmolality, osmolarity, viscosity, clarity, color, tonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the primary formulations and / or polypeptide and / or protein.

The formulations will, of course, depend on, for example, the particular protein to be formulated, the different active ingredients, such as other pharmaceuticals, that will be included in the formulation, the intended route of administration, the method of administration to be employed, the dosage, dosage frequency and delivery format, among others.

Formulations according to some of the preferred embodiments in various aspects of the invention provide compositions comprising a protein, preferably a pharmaceutical protein, and a solvent, the protein having a buffering capacity per unit volume of at least about: the buffer 4.0 mM sodium acetate as determined in the range of pH 5.0 to 4.0 or pH 5.0 to 5.5 as described in Example 1 or 2 and elsewhere herein.

Formulations in accordance with some of the preferred embodiments in various aspects of the invention provide self-buffering protein compositions, particularly pharmaceutical protein compositions, wherein, excluding the buffering capacity of the protein, the buffering capacity per unit volume of the composition is equal to or less than that of 1.0 or 1.5 or 2.0 mM sodium acetate buffer as determined in the range of pH 5.0 to 4.0 or pH 5.0 to 5.5 as described in Example 1 or 2 and elsewhere herein.

The formulations according to some of the preferred embodiments in various aspects of the disclosure provide self-buffering protein compositions, particularly protein pharmaceutical compositions, comprising a protein and a solvent, wherein at the pH of the composition, the buffering capacity of the protein is at least approximately: 1.00 or 1.50 or 1.63 or 2.00 or 3.00 or 4.00 or 5.00 or 6.50 or 8.00 or 10.0 or 15.0 or 20 , 0 or, 0 or 40.0 or 50.0 or 75.0 or 100 or 125 or 150 or 200 or 250 or 300 or 350 or 400 or 500 or 700 or 1,000 or 1,500 or 2,000 or 2,500 or 3,000 or 4,000 or 5,000 mEq per liter and per pH change of one pH unit.

Formulations according to some of the preferred embodiments in various aspects of the invention provide self-buffering protein compositions, particularly protein pharmaceutical compositions, comprising a protein and a solvent, wherein at the pH of the composition, excluding protein, the buffer capacity per unit volume of the composition is equal to or less than that of a 0.50 or 1.00 or 1.50 or 2.00 mM acetate buffer as determined in the range of pH 5.0 to 4.0 or pH 5.0 to 5.5 as described in Example 1 or 2 and elsewhere in this document.

Formulations according to some of the preferred embodiments in various aspects of the invention provide self-buffering protein compositions, particularly protein pharmaceutical compositions, comprising a protein and a solvent, wherein at the desired pH the protein provides at least about 80 % of the buffer capacity of the composition.

Formulations according to some of the preferred embodiments in various aspects of the invention provide self-buffering protein compositions, particularly protein pharmaceutical compositions, comprising a protein and a solvent, A, wherein the concentration of the protein is between about: 20 and 400, or 20 and 300, or 20 and 250, or 20 and 200, or 20 and 150 mg / ml.

Formulations according to some of the preferred embodiments in various aspects of the invention provide self-buffering protein compositions, particularly protein pharmaceutical compositions, comprising a protein and a solvent, wherein the pH maintained by the buffering action of the protein is a pH between approximately: 3.5 and 8.0, or 4.0 and 6.0, or 4.0 and 5.5, or 4.5 and 5.5.

Formulations according to some of the preferred embodiments in various aspects of the invention provide self-buffering protein compositions, particularly protein pharmaceutical compositions, comprising a protein and a solvent, wherein the salt concentration is less than: 150 mM or 125 mM or 100 mM or 75 mM or 50 mM or 25 mM.

Formulations according to some of the preferred embodiments in various aspects of the invention provide self-buffering protein compositions, particularly protein pharmaceutical compositions, comprising a protein and a solvent, and further comprising one or more pharmaceutically acceptable salts; osmotic balancing agents (tonicity agents); antioxidants; antibiotics; antifungals; freight forwarders; lyoprotectants; antifoaming agents; chelating agents; preservatives; colorants; analgesics; or additional pharmaceutical agents.

Formulations according to some of the preferred embodiments in various aspects of the invention provide self-buffering protein compositions, particularly protein pharmaceutical compositions, comprising a protein and a solvent, and further comprising one or more pharmaceutically acceptable polyols in an amount that is hypotonic, isotonic or hypertonic, preferably approximately isotonic, particularly and preferably isotonic, especially preferably one or more of sorbitol, mannitol, sucrose, trehalose or glycerol, particularly especially and preferably approximately 5% sorbitol, 5% mannitol, sucrose 9%, 9% trehalose or 2.5% glycerol, very especially in this connection 5% sorbitol, 5% mannitol, 9% sucrose, 9% trehalose or 2.5% glycerol.

Formulations according to some of the preferred embodiments in various aspects of the invention provide self-buffering protein compositions, particularly protein pharmaceutical compositions, comprising a protein and a solvent, and further comprising one or more pharmaceutically acceptable surfactants, preferably one or more plus polysorbate 20, polysorbate 80, other sorbitan fatty acid esters, polyethoxylates and poloxamer 188, particularly preferably polysorbate 20 or polysorbate 80, preferably about 0.001 to 0.1% polysorbate 20 or polysorbate 80, most preferably about 0.002 to 0.02% polysorbate 20 or polysorbate 80, especially 0.002 to 0.02% polysorbate 20 or polysorbate 80.

Formulations according to some of the preferred embodiments in various aspects of the invention provide self-buffering protein compositions, particularly protein pharmaceutical compositions, comprising a protein and a solvent, wherein the protein is a pharmaceutical agent and the composition is a formulation. sterile thereof suitable for the treatment of a human or veterinary medical subject.

Also among formulations in accordance with various aspects and embodiments of the invention described herein are lyophilized compositions in accordance with the foregoing, particularly lyophilized compositions which when reconstituted provide a formulation as described above and elsewhere herein. document.

to. Excipients and other Additional Ingredients

As discussed above, certain embodiments in accordance with aspects of the invention provide self-buffering protein compositions, particularly protein pharmaceutical compositions, which they comprise, in addition to protein, particularly a pharmaceutical protein, one or more excipients such as those illustratively described in this section and elsewhere herein. Excipients can be used in the invention in this regard for a wide variety of purposes, such as adjusting the physical, chemical, or biological properties of formulations, such as adjusting viscosity, and / or the processes of the invention to improve the efficacy and / or stabilization of such formulations and processes against degradation and deterioration due to, for example, stresses that occur during manufacture, transportation, storage, pre-use preparation, administration and thereafter.

There are a variety of disclosures available on protein stabilization and useful formulation materials and methods in this regard, such as Arakawa et al., "Solvent interactions in pharmaceutical formulations," Pharm Res. 8 (3): 285-91 (1991) ; Kendrick et al., "Physical stabilization of proteins in aqueous solution," in: RATIONAL DESIGN OF STABLE PROTEIN FORMULATIONS: THEORY AND PRACTICE, Carpenter and Manning, eds. Pharmaceutical Biotechnology. 13: 61-84 (2002) and Randolph et al., "Surfactant-protein interactions," Pharm Biotechnol. 13: 159-75 (2002), particularly in parts related to excipients and processes thereof for formulations of self-buffering proteins according to the present invention, especially as regards pharmaceutical products and processes of proteins for veterinary and / or human medical uses .

Various excipients useful in the invention are listed in Table 1 and are further described below.

Table 1: Types of Exci ientes Their Functions

continuation

i. You go out

The salts can be used in accordance with certain of the preferred embodiments of the invention to, for example, adjust the ionic strength and / or isotonicity of a self-buffering formulation and / or improve the solubility and / or physical stability of a self-buffering protein. or another ingredient of a self-buffering protein composition according to the invention.

As is well known, ions can stabilize the natural state of proteins by binding to charged moieties on the surface of the protein and protecting the charged and polar groups on the protein and reducing the strength of their electrostatic interactions, attractive and repulsive interactions. Ions can also stabilize the denatured state of a protein by binding, in particular, to denatured peptide bonds (-CONH) of the protein. Likewise, ionic interaction with charged and polar groups on a protein can also reduce intermolecular electrostatic interactions and thus prevent or reduce aggregation and insolubility of proteins.

Ionic species differ significantly in their effects on proteins. A series of categorical classifications of ions and their effects on proteins have been developed that can be used in the formulation of self-buffering protein compositions in accordance with the invention. An example is the Hofmeister series, which classifies ionic and polar nonionic solutes by their effect on the conformational stability of proteins in solution. Stabilizing solutes are called "cosmotropic". The destabilizing solutes are called chaotropic. Cosmotropic agents are commonly used in high concentrations (eg,> 1 molar ammonium sulfate) to precipitate proteins from solution ("salt addition protein precipitation"). Chaotropic agents are commonly used to denature and / or solubilize proteins ("dissolving proteins by adding salt"). The relative efficiency of ions for "salt addition protein precipitation and salt protein dissolution" defines their position in the Hofmeister series.

In addition to their utilities and drawbacks (as discussed above), salts are also effective in reducing the viscosity of protein formulations and can be used in the invention for that purpose.

In order to maintain isotonicity in a precursor formulation according to the preferred embodiments of the invention, improve the solubility and / or stability of the proteins, improve the viscosity characteristics, avoid detrimental effects of the salt on the stability and aggregation of protein, and to prevent salt-mediated degradation of proteins, the salt concentration in self-buffering formulations according to various preferred embodiments of the invention is less than 150 mM (for monovalent ions) and 150 mEq / liter for ions multivalent. In this regard, in certain particularly preferred embodiments of the invention, the total salt concentration is from about 75 mEq / L to about 140 mEq / L.

ii. Amino acids

Free amino acids can be used in protein formulations in accordance with various preferred embodiments of the invention such as, to name a few, bulking agents, stabilizers and antioxidants. However, the amino acids comprised in the self-buffering protein formulations according to the invention do not provide buffering action. For this reason, those with significant buffering capacity are not used, they are not used at any pH around which they have significant buffering activity, or they are used at low concentration so that, as a result, their buffering capacity in the formulation is not significant. . This is particularly the case for histidine and other amino acids that are commonly used as buffers in pharmaceutical formulations.

Subject to the above consideration, lysine, proline, serine, and alanine can be used to stabilize proteins in a formulation. Glycine is useful in lyophilization to ensure the correct structure and properties of the crust. As a result, it is a common ingredient in reconstituted lyophilized and lyophilized formulations, such as Neumega®, Genotropin®, and Humatrope®. Arginine can be useful to inhibit protein aggregation, both in liquid and lyophilized formulations, such as Activase®, Avonex®, and liquid Enbrel®. Methionine is useful as an antioxidant.

iii. Polyols

Polyols include sugars, eg, mannitol, sucrose, and sorbitol, and polyhydric alcohols such as, eg, glycerol and propylene glycol, and, for purposes of discussion herein, polyethylene glycol (PEG) and related substances. Polyols are cosmotropic. They are useful stabilizing agents in both liquid and lyophilized formulations to protect proteins from physical and chemical degradation processes. Polyols are also useful for adjusting the tonicity of formulations.

Among the polyols useful in the invention in this regard is mannitol, commonly used to ensure structural stability of the crust in lyophilized formulations, such as, for example, Leukine®, Enbrel®-Lyo and Betaseron®. Ensures structural stability to the scab. It is generally used with a lyoprotectant, for example sucrose. Sorbitol and sucrose are among the preferred agents for adjusting tonicity and as stabilizers to protect against freeze-thaw stresses during shipping or batch preparation during the manufacturing process. Reducing sugars (containing free aldehyde or ketone groups), such as glucose and lactose, can glycosylate lysine and arginine residues on the surface. Therefore, they are generally not among the preferred polyols for use in accordance with the invention. Furthermore, the sugars that form such reactive species, such as sucrose, which hydrolyze to fructose and glucose under acidic conditions, and consequently generate glycosylation, are also not among the preferred amino acids of the invention in this regard. PEG is useful for stabilizing proteins and as a cryoprotectant and can be used in the invention in this regard, as it is in Recombinate®.

iv. Surfactants

Protein molecules are susceptible to adsorption on surfaces and denaturation and consequent aggregation at air-liquid, solid-liquid, and liquid-liquid interfaces. These effects generally scale inversely with protein concentration. These harmful interactions generally scale inversely with protein concentration and are generally exacerbated by physical agitation, such as that generated during transportation and handling of a product.

Surfactants are commonly used to prevent, minimize or reduce adsorption on the surface. Surfactants useful in the invention in this regard include polysorbate 20, polysorbate 80, other fatty acid esters of sorbitan polyethoxylates, and poloxamer 188.

Surfactants are also commonly used to control the conformational stability of proteins. The use of surfactants in this regard is protein specific in that any given surfactant will generally stabilize some proteins and destabilize others.

Polysorbates are susceptible to oxidative degradation and often, as supplied, contain sufficient amounts of peroxides to cause oxidation of the side chains of protein moieties, especially methionine. Consequently, polysorbates must be used with care, and when used, they must be used at their lowest effective concentration. In this regard, polysorbates exemplify the general rule that excipients should be used in their lowest effective concentrations.

v. Antioxidants

A variety of processes can result in the harmful oxidation of proteins in pharmaceutical formulations. To some extent, damaging oxidation of proteins in pharmaceutical formulations can be prevented by maintaining adequate oxygen levels and room temperature and avoiding exposure to light. Antioxidant excipients can also be used to prevent oxidative degradation of proteins. Antioxidants useful in this regard include reducing agents, oxygen / free radical scavengers, and chelating agents. Antioxidants for use in therapeutic protein formulations according to the invention are preferably water soluble and maintain their activity for the shelf life of a product. EDTA is a preferred antioxidant according to the invention in this regard and can be used in the invention in the same manner as it has been used in fibroblast acid growth factor formulations and in products such as Kineret® and Ontak®.

Antioxidants can damage proteins. For example, reducing agents, such as glutathione, in particular, can break intramolecular disulfide bonds. Therefore, antioxidants for use in the invention are selected to, among other things, eliminate or sufficiently reduce their possibility of damaging proteins in the formulation.

saw. Metal Ions

Formulations according to the invention may include metal ions which are protein cofactors and which are necessary to form protein coordination complexes, such as the zinc necessary to form certain insulin suspensions. Metal ions can also inhibit some processes that break down proteins. However, metal ions can also catalyze physical and chemical processes that degrade proteins.

Magnesium ions (10-120 mM) can be used to inhibit the isomerization of aspartic acid to isoaspartic acid. Ca 2 ions (up to 100 mM) can increase the stability of human deoxyribonuclease (rhDNase, Pulmozyme®). Mg + 2, Mn + 2, and Zn + 2, however, can stabilize rhDNase. Similarly, Ca + 2 and Sr + 2 can stabilize Factor VIII, which can be destabilized by Mg2 +, Mn + 2 and Zn + 2, Cu + 2 and Fe + 2, and its aggregation can be increased by Al + ions. 3.

vii. Preservatives

Preservatives are necessary when developing multi-dose parenteral formulations that involve more than one draw from the same container. Its main function is to inhibit microbial growth and ensure the sterility of the product during the shelf life or duration of use of the drug product. Commonly used preservatives include benzyl alcohol, phenol, and m-cresol. Although preservatives have a long history of use with small molecule parenterals, developing protein formulations that include preservatives can be challenging. Preservatives almost always have a destabilizing (aggregating) effect on proteins, and this has become an important factor in limiting their use in multidose protein formulations. To date, most protein drugs have been formulated for single use only. However, when multi-dose formulations are possible, they have the additional advantage of allowing patient convenience and greater marketability. A good example is human growth hormone (hGH), where the development of preserved formulations has led to the commercialization of more convenient and multipurpose injection pen presentations. At least four of these injection pen devices containing preserved hGH formulations are currently commercially available. Norditropin® (liquid, Novo Nordisk), Nutropin AQ® (liquid, Genentech) & Genotropin (lyophilized - double chamber cartridge, Pharmacia & Upjohn) contains phenol while Somatrope® (Eli Lilly) is formulated with m-cresol.

Several aspects must be considered during the formulation and development of preserved dosage forms. The effective concentration of preservatives in the drug product must be optimized. This requires testing a given preservative in dosage form with concentration ranges that confer antimicrobial efficacy without compromising protein stability. For example, three preservatives were successfully explored in the development of a liquid formulation for the interleukin-1 receptor (Type I) using differential scanning calorimetry (DSC). Preservatives were ranked based on their impact on stability at concentrations commonly used in marketed products.

As might be expected, the development of liquid formulations containing preservatives is more difficult than lyophilized formulations. Lyophilized products can be lyophilized without the preservative and reconstituted with a diluent containing preservative at the time of use. This shortens the time a preservative is in contact with the protein, significantly minimizing the associated stability risks. With liquid formulations, the efficacy and stability of the preservative must be maintained throughout the shelf life of the product (~ 18 to 24 months). An important point to keep in mind is that the efficacy of the preservative must be demonstrated in the final formulation containing the active drug and all the excipient components.

Self-buffering protein formulations according to the invention, particularly self-buffering biopharmaceutical protein formulations, will generally be designed for specific routes and methods of administration, for specific doses of administration and frequencies of administration, for specific treatments of specific diseases, with intervals of bioavailability and persistence, among other things.

Therefore, the formulations can be designed according to the invention for delivery by any suitable route, including, but not limited to, oral, auditory, ophthalmic, rectal and vaginal, and parenterally, including intravenous and intra-arterial injection, intramuscular injection. and subcutaneous injection.

b. Formulations for Parenteral Administration

Formulations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions and suspensions can be prepared from sterile powders or granules using one or more of the carriers or diluents mentioned for use in formulations for oral administration or by using other suitable dispersing or wetting agents and suspending agents.

When parenteral administration is contemplated, therapeutic compositions for use in this invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired protein in a pharmaceutically acceptable carrier. A particularly suitable vehicle for parenteral injection is sterile pure water in which the protein is formulated as a sterile self-buffering isotonic solution.

Such preparations may also involve formulating the desired protein in the form of, among other things, injectable microspheres, bioerodible particles, polymeric compounds (polylactic acid, polyglycolic acid), beads or liposomes, including those that provide sustained release or control. Such formulations can be introduced by implantable drug delivery devices, among others.

Formulations for parenteral administration may also contain substances that adjust viscosity, such as carboxymethylcellulose, sorbitol, and dextran. The formulations may also contain ingredients that increase the solubility of the desired protein or other ingredients and those that stabilize one or more of such ingredients, including in some cases, self-buffering protein.

c. Formulations for Pulmonary Administration

A pharmaceutical composition according to certain embodiments of the invention may be suitable for inhalation. For pulmonary administration, the pharmaceutical composition can be administered in the form of an aerosol or with an inhaler that includes a dry powder aerosol. For example, a binding agent can be formulated as a dry powder for inhalation. Inhalation solutions can also be formulated with an aerosol delivery propellant. In yet another embodiment, the solutions can be nebulized. Pulmonary administration is further described in PCT Application No. PCT / US94 / 001875, which describes the pulmonary delivery of chemically modified proteins.

c. Formulations for Oral Administration

For oral administration, the pharmaceutical composition can be in the form of, for example, a tablet, capsule, suspension or liquid. The pharmaceutical composition is preferably prepared in the form of a dosage unit containing a particular amount of the active ingredient. Examples of such dosage units are tablets or capsules. Formulations for oral administration in accordance with the invention in this regard can be conventionally prepared wherein the self-buffering protein provides the buffering in the formulation as described elsewhere herein.

and. Controlled Release Formulations

Among additional formulations that may be useful in the invention as described herein are sustained and controlled delivery formulations. Techniques for preparing such sustained and controlled delivery formulations that can be used in accordance with various aspects and preferred embodiments of the invention are well known to those skilled in the art. Among these are delivery methods that use liposome carriers, bioerodible microparticles, porous beads, and semipermeable polymer matrices, such as those described in PCT / US93 / 00829; US 3,773,919; EP 58,481; Sidman et al., Biopolymers, 22: 547-556 (1983); Langer et al., J. Biomed. Mater. Res., 15: 167-277, (1981); Langer et al., Chem. Tech., 12: 98-105 (1982); EP 133,988; Eppstein et al., Proc. Natl. Acad. Sci. (USA), 82: 3688-3692 (1985); e P 36,676; EP 88,046; and EP 143,949, particularly in parts related to controlled and sustained delivery pharmaceutical protein formulations of autotamponants according to the invention described herein.

F. Sterilization

The pharmaceutical composition to be used for in vivo administration must generally be sterile. This can be accomplished by filtration through sterile filter membranes. When the composition is lyophilized, sterilization using this method can be performed either before or after lyophilization and reconstitution. The composition for parenteral administration can be stored in lyophilized form or in solution. In addition, parenteral compositions are generally placed in a container that has a sterile access port, for example, an intravenous solution bag or vial that has a cap pierceable by a hypodermic injection needle.

g. Storage

Once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or a dehydrated or lyophilized powder. Such formulations can be stored in a ready-to-use form or in a form (eg, lyophilized) that is reconstituted prior to administration.

h. Additional Pharmaceutical Agents

The self-buffering protein compositions according to the invention, particularly the self-buffering pharmaceutical protein compositions, may comprise in addition to the self-buffering protein of the composition, one or more additional pharmaceutical agents. Said agents can also be proteins, or they can be other types of agents. Such agents include those for the prevention or treatment of any disorder or disease. Such agents include, for example, antibiotics and antifungals. They also include agents for treating human disorders, including but not limited to, agents for treating inflammatory diseases, cancers, metabolic disorders, neurological and kidney disorders, to name just a few. Agents that can be used in the invention in this regard also include agents useful for enhancing the action of a self-buffering composition and / or preventing, ameliorating, or treating any undesirable side effects of administration thereof.

i. Methods for Preparing Self-Buffering Protein Formulations

Compositions according to the invention can be produced using standard, well known methods for preparing, formulating and using proteins, particularly pharmaceutical proteins. In some of the preferred embodiments of various aspects of the invention in this regard, the methods for preparing the compositions comprise the use of counter ions to remove residual buffering agents. In this regard, the term "counter ion" is any polar or charged constituent that acts to displace the buffer from the composition during its preparation. Useful counter ions in this regard include, for example, glycine, chloride, sulfate, and phosphate. The term counterion in this regard is used to refer to the same as displacement ion.

Residual buffering agents can be removed using the counter ions in this regard, using a variety of well known methods, including but not limited to conventional dialysis methods and high throughput membrane diffusion based methods such as tangential flow diafiltration. Methods for the removal of residual buffer employing a counter ion in this regard can also, in some cases, be carried out using size exclusion chromatography.

In certain preferred embodiments related in this regard, the compositions according to the invention are prepared by a process involving dialysis against a solution without buffer at a pH lower than that of the preparation containing the self-buffering protein. In particularly preferred embodiments of the invention in this regard, the buffer-free solution comprises counter ions, particularly those that facilitate the removal of residual buffer and do not adversely affect the self-buffering protein or formulation thereof. In further particularly preferred embodiments of the invention in this regard, after dialysis, the pH of the preparation is adjusted to the desired pH using dilute acid or dilute base.

In certain preferred embodiments particularly related in this regard, the compositions according to the invention are prepared by a process involving diafiltration against a solution without buffer at a pH lower than that of the preparation containing the self-buffering protein. In particularly preferred embodiments of the invention in this regard, the buffer-free solution comprises counter ions, particularly those that facilitate the removal of residual buffer and do not adversely affect the self-buffering protein or formulation thereof. In further particularly preferred embodiments of the invention in this regard, after diafiltration, the pH of the preparation is adjusted to the desired pH using dilute acid or dilute base.

5. Routes of administration

Formulations according to the invention, in various embodiments, can be administered by a variety of suitable routes, well known to those skilled in the art of administering therapeutic agents to a subject. In embodiments of the invention in this regard, one or more formulations, as described elsewhere herein, are administered via the gastrointestinal tract. In other embodiments, one or more formulations such as those described elsewhere in this document are administered parenterally. In various embodiments, one or more formulations can be administered via the gastrointestinal tract in conjunction with one or more different formulations administered parenterally.

Such routes in a variety of embodiments include, but are not limited to, administration of the compositions orally, ocularly, mucosa, topical, rectal, pulmonary, such as by inhalation spray and epicutaneous. The following parenteral routes of administration are also useful in various embodiments of the invention: administration by intravenous, intraarterial, intracardiac, intraspinal, intrathecal, intraosseous, intraarticular, intrasynovial, intracutaneous, intradermal, subcutaneous, peritoneal, and / or intramuscular injection. In some embodiments, intravenous, intraarterial, intracutaneous, intradermal, subcutaneous, and / or intramuscular injections are used. In some embodiments, intravenous, intraarterial, intracutaneous, subcutaneous, and / or intramuscular injections are used.

In certain embodiments of the invention, the compositions are administered locally, for example, by intraocular injection to treat ocular neovascularization, retinopathy, or age-related macular degeneration.

6. Dose

The amount of a self-buffering protein formulation administered and the dosage regimen to treat a condition with the formulation depends on a variety of factors, including the subject's age, weight, sex and medical condition, type of disease, severity of disease, route and frequency of administration, and particular formulation employed. In particular, the amount will depend on the therapeutic protein to be administered and any other therapeutic agent to be administered in conjunction with it. Doses can be determined for formulations according to the invention using well established standard pharmaceutical procedures for this purpose.

7. Dosage Guidelines

The formulations of the invention can be administered in dosages and by techniques well known to those skilled in the medical and veterinary art taking into account factors such as the age, sex, weight and condition of the particular patient, and the formulation to be administered. (eg, solid vs. liquid). Doses for humans or other mammals can be determined without undue experimentation by one of ordinary skill in the art, from this disclosure, the documents cited herein, and knowledge in the art.

According to various embodiments, appropriate dosages and dosage schedules will depend on numerous factors and may vary in different circumstances. The parameters that will determine the optimal dosage schedules to be administered will typically include some or all of the following: the disease to be treated and its phase; the subject's species, health, gender, age, weight, and metabolic rate; other treatments that are being administered; and the possible complications expected by the history or genotype of the subject.

The optimal dosing schedule in a given situation will also take into consideration the nature of the formulation, the way it is administered, the route of distribution after administration, and the rate at which both the sites of action and the site of action will be cleared. subject's body. Finally, determining the optimal dosage will preferably provide an effective dose that is neither below the threshold of maximum benefit nor above the threshold at which the detrimental effects associated with the dose of the active ingredients outweigh the benefits of augmentation. of the dose.

It will be appreciated that a "dose" may be delivered all at once, fractionally, or continuously over a period of time. The full dose can also be given in one place or divided into several places. Also, the doses may remain the same during a treatment, or they may vary.

In various embodiments, the formulations according to the invention are administered in an initial dose and, thereafter, are maintained by additional administrations. A formulation of the invention in some embodiments is administered by one method initially, and then administered by the same method or by one or more different methods. The doses of the current administrations can be adjusted to maintain the levels of the active principles in the subject at certain values. In some embodiments, the compositions are administered initially, and / or to maintain their level in the subject, by intravenous injection. In a variety of embodiments, other forms of administration are used.

The formulations of the invention can be administered at many frequencies over a wide time range, including any suitable frequency and time range that provides an effective dose for treatment. Doses can be given continuously, given every few hours, one or more times a day, every day, every other day, or several times a week, or less frequently. In some embodiments, they are administered for periods of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more days. In some embodiments, they are administered for periods of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more months. In a variety of embodiments, they are administered for periods of one, two, three, four, five, six, seven, eight, nine, ten or more years. Appropriate schedules for initial administration and additional doses for sequential administrations may all be the same or they may be variable. Appropriate guidelines may be established by one of ordinary skill in the art, based on this disclosure, the documents cited herein, and knowledge in the art. In general, the treatment durations will be proportional to the duration of the disease process, the efficacy of the treatments being applied, and the status and response of the subject being treated.

8. Diseases and Treatments

The self-buffering pharmaceutical protein compositions according to the invention, in preferred embodiments, are for use in treating subjects suffering from a wide variety of disorders and diseases. As noted elsewhere herein, the invention provides, among others, self-buffering pharmaceutical antibody compositions, antibody-derived pharmaceutical proteins, and antibody-related pharmaceutical proteins, which may comprise Fc effector functions and binding domains specific for a wide variety of targets related to disease and useful for treating disease. These proteins and the self-buffering compositions thereof are described in detail herein above, as well as their use in the treatment of various disorders and diseases associated with their targets. Methods for using the compositions, including formulation methods, administration methods, dosages, and dosing methods are fully described in an illustrative manner above. The formulation and administration of any particular composition of the invention can be adapted to the treatment of a particular disease, using techniques known and common in the art to do so, in light of the guidance provided by the present description of the invention. Among the diseases usefully treated using self-buffering pharmaceutical protein formulations in accordance with various aspects and preferred embodiments of the invention are inflammatory diseases, cancers, metabolic disorders, neurological and kidney disorders, to name just a few.

9. Packaging and Kits

The invention also provides kits comprising self-buffering protein formulations, particularly kits comprising in one or more packages, a self-buffering pharmaceutical protein formulation and instructions regarding the use thereof, particularly such kits wherein the formulation is a pharmaceutically acceptable formulation. for human use. Among the preferred kits are those that comprise one or more packages of a self-buffering protein formulation of the invention and one or more separate documents, information related to the content of the kit and / or the use of its content, particularly those in which the protein is a biopharmaceutical protein, especially those in which the protein is a biopharmaceutical protein formulated for the treatment of disease in humans.

In certain aspects of the invention in this regard, preferred kits include kits such as those above that further comprise one or more one or more chamber syringes (eg, liquid syringes and lyosyringes) for administering one or more self-buffering protein formulations of the invention. In certain aspects of the invention in this regard, some of the particularly preferred kits further comprise pre-filled syringes. In particularly further preferred embodiments in this regard, the kits comprise a self-buffering pharmaceutical composition for parenteral administration, sealed in a partial vacuum vial in a form ready to load into a syringe and administer to a subject. In especially preferred embodiments in this regard, the composition is arranged therein under partial vacuum. In all of these aspects and in others, in certain further particularly preferred embodiments, the kits contain one or more vials according to any of the foregoing, wherein each vial contains a single unit dose for administration to a subject. In all these aspects and others, the invention further relates to kits comprising lyophilisates, arranged as above, which after reconstitution provide compositions according thereto. In this regard, the invention further provides, in some of its preferred embodiments, kits containing a lyophilizate according to the invention and a sterile diluent to reconstitute the lyophilizate.

Examples

The present invention is further described by means of the following non-limiting illustrative Examples.

Example 1: Acid Titrations and Buffer Capacities of Sodium Acetate Buffers in the Range of pH 5.0 to 4.0

A stock solution of known acetic acid concentration was prepared by diluting ultra-pure glacial acetic acid in HPLC grade water and then titrating the pH to the desired value with NaOH. The reserves were equilibrated in air and at 21 ° C. Volumetric standards were prepared at a concentration of 1 N and diluted as necessary with HPLC water.

One mM, 2.5 mM, 5 mM, 7.5 mM, 10 mM, and 15 mM sodium acetate buffers were prepared by diluting the stock in HPLC water. The solutions were titrated with HCl. 0.2N HCl was used for the 1, 2.5 and 5 mM solutions, 0.4 N HCl for the 7.5 mM solution and 0.8 N HCl was used for the 10 and 15 mM solutions. Titrations were performed using standard laboratory analytical techniques.

Figure 1, Panel A shows the titer data and the least squares trend lines calculated from the data for each solution. The slope of the trend line calculated from each data set was taken as the buffer capacity of the corresponding acetate buffer. The linear dependence of the buffer capacity with the concentration of the acetate buffer is shown in Figure 1, Panel B.

Example 2: Base Titrations and Buffer Capacities of Sodium Acetate Buffers in the Range of pH 5.0 to 5.5

Acetate buffer stocks and solutions for titration were prepared as described in Example 1. The solutions were titrated as described in Example 1, except that the solutions were titrated from pH 5.0 to 5.5 and the titrations were performed using NaOH instead of HCl. 0.2 N NaOH was used to titrate the 1, 2.5 and 5 mM solutions and 0.4 N NaOH was used for the 7.5, 10 and 15 mM solutions. The results of the titrations are shown in Figure 2A. The linear dependence of the buffer capacity with the acetate buffer concentration is shown in Figure 2B.

Example 3: Determination of Acetate by HPLC

Acetate in acetate buffer samples was determined using analytical SE-HPLC. A standard curve for maximum areas as a function of acetate concentration was established by analysis of acetate in buffers of known acetate concentration. The amount of acetate in the test samples was interpolated from the standard curve. A standard curve is shown in Figure 3. The nominal and measured amount of acetate in the test buffers is tabulated below the standard curve in the figure.

Example 4: Acid Titrations of Ac-hOPGL Formulations in the Range from pH 5.0 to pH 4.0

Bulk Ac-hOPGL was diafiltered into 10 mM acetate (nominal value), 5% sorbitol, pH 5.0 against 5.25% sorbitol, pH 3.2 (adjusted with HCl) in a LABSCALE TFF® system (Millipore ) with a multifilter cassette, using 3 Millipore Pellicon XL 50 regenerated cellulose ultrafiltration membranes. The diafiltration solution was exchanged 8 to 10 times during the course of diafiltration for each formulation. After diafiltration, the pH of the resulting unbuffered solution was measured and adjusted to pH 5.0, using 0.05 N HCl or 0.05 N NaOH.

Solutions of one, 10, 30, 60, 90, and 110 mg / ml were prepared for titration by dilution. The pH of each dilution was adjusted to pH 5.0 with NaOH or HCl as necessary. Titrations were carried out as described in the previous Examples. 0.2 N HCl was used to titrate the 1, 10, and 30 mg / ml solutions. 0.4N HCl was used to titrate the 60 mg / ml solution. 0.8 N HCl was used to titrate the 90 and 110 mg / ml solutions.

The results of the titrations are represented in Figure 4. The least squares regression line is shown for the data set for each concentration. The buffer capacity was taken as the slope of the regression line for each concentration.

Example 5: Base Titrations of Ac-hOPGL Formulations in the Range from pH 5.0 to pH 6.0

Solutions of one, 10, 30, 60, 90, and 110 mg / ml Ac.hOPGL were prepared for titration as described in Example 4. Base titrations were carried out using NaOH as described in previous Examples. 0.2 N NaOH was used for the 1, 10, 30 and 60 mg / ml solutions and 0.4 N NaOH was used for the 90 and 110 mg / ml solutions.

The results of the titrations are plotted in Figure 5. Linear regression lines are shown for the data for each concentration. The buffer capacity was taken as the slope of the regression line for each concentration.

Example 6: Residual Acetate Levels in Self-buffering Ac-hOPGL Formulations

The amount of residual acetate was determined in Ac-hOPGL formulations using the methods described in Example 3. The results are graphically represented in Figure 6, which shows a standard curve that relates HPLC measurements to acetate concentrations and, below the graph, a tabulation of the results of the determinations made on Ac-hOPGL formulations at different concentrations. The Ac-hOPGL concentrations are indicated on the left ("Nominal") and the measured concentration of acetate in each of the Ac-hOPGL concentrations is indicated on the right.

Example 7: Buffer capacity of Ac-hOPGL Formulations Plus or Minus Acetate Residual in the Range of pH 5.0 to 4.0

Self-buffered Ac-hOPGL formulations were prepared and titrated with HCl as described in the Examples above. Furthermore, the data was adjusted by subtracting the contribution of residual acetate buffer based on the determination of acetate content by SE-HPLC as described in, for example, Example 3. Buffer capacities were determined as described above. The same analysis was performed on both data sets. The results, represented in Figure 7, show the effect of residual acetate on the buffering capacity of Ac-hOPGL preparations. The results make it clear that the residual acetate buffering capacity is a minor factor in the buffering capacity of the self-buffering Ac-hOPGL formulations that were tested.

Example 8: Ac-hOPGL Buffer Capacity Plus or Minus Residual Acetate in the Range of pH 5.0 to 6.0

Self-buffered Ac-hOPGL formulations were prepared and titrated with NaOH as described in the Examples above. Furthermore, the data was adjusted by subtracting the contribution of residual acetate buffer based on the determination of acetate content by SE-HPLC as described in, for example, Example 3. Buffer capacities were determined as described above. The same analysis was performed on both data sets. The results, represented in Figure 8, show the effect of residual acetate on the buffering capacity of Ac-hOPGL preparations. The results make it clear that the residual acetate buffering capacity is a minor factor in the buffering capacity of the self-buffering Ac-hOPGL formulations that were tested.

Example 9: pH and Stability of Ac-hOPGL in Self-Buffed and Conventionally Buffered Formulations

Self-buffering formulations of Ac-hOPGL were prepared as described in the previous Examples. In addition, formulations containing a conventional buffering agent, either acetate or glutamate, were prepared. All formulations contained 60 mg / ml Ac-hOPGL. The stability of pH and Ac-hOPGL in the formulations was monitored during six months of storage at 4 ° C. Stability was monitored by determining monomeric Ac-hOPGL in the formulations over the course of storage time. The determination was carried out using SE-HPLC as described above. The results for the three formulations are shown in Figure 9. Panel A shows the stability of Ac-hOPGL in the three formulations. Stability in the self-buffered formulation is as good as in conventionally buffered formulations. Panel B shows the pH stability of the three formulations. Again, the pH stability in the self-buffered formulation is as good as in conventionally buffered formulations.

Reference Example 1: Titration and Buffer Capacities of Ac-hB7RP1 - pH 5.0 to 4.0

Self-buffering formulations of Ac-hB7RP1 were prepared at concentrations of 1, 10, 30 and 60 mg / ml, as described for Ac-hOPGL in the previous Examples. Titrations were carried out using HCl as described above. Furthermore, the data was adjusted by subtracting the contribution of the residual acetate buffer based on the determination of the acetate content by SE-HPLC as described in, for example, Example 3. Figure 10, panel A shows the results of the titrations . Figure 10, panel B shows the dependence of the buffer capacity with the concentration of the Ac-hB7RP1 formulations before and after subtracting the contribution of the residual acetate buffer. The results clearly show the self-buffering capacity of Ac-hB7RP1 in this pH range. At 40 mg / ml, it provides approximately the same buffer capacity in this pH range as 10 mM sodium acetate buffer. At 60 mg / ml, it provides approximately the same buffer capacity as 15 mM sodium acetate buffer.

Reference Example 21: Titration and Buffer Capacities of Ac-hB7RP1 - pH 5.0 to 6.0

Self-buffering formulations of Ac-hB7RP1 were prepared at concentrations of 1, 10, 30 and 60 mg / ml, as described for Ac-hOPGL in the previous Examples. Titrations were carried out using NaOH as described above. Furthermore, the data was adjusted by subtracting the contribution of the residual acetate buffer based on the determination of the acetate content by SE-HPLC as described in, for example, Example 3. Figure 11, panel A shows the results of the titrations . Figure 11, panel B shows the dependence of the buffer capacity with the concentration of the Ac-hB7RP1 formulations before and after subtracting the contribution of the residual acetate buffer. The results clearly show the self-buffering capacity of Ac-hB7RP1 in this pH range. At 60 mg / ml, it provides approximately the same buffer capacity in this pH range as 10 mM sodium acetate buffer.

Reference Example 3: Stability of Ac-hB7RP1 in Self-Buffering and Conventionally Buffered Formulations at 4 ° C and 29 ° C

Ac-hB7RP1 was prepared as described in the Reference Examples above and formulated as described above, in self-buffering formulations and in formulations using a conventional buffering agent, either acetate or glutamate. All formulations contained 60 mg / ml of Ac-hB7RP1. The stability of the pH of the solutions and of Ac-hB7RP1 in the solution was monitored during 26 months of storage at 4 ° C or 29 ° C. Stability was controlled by determining monomeric Ac-hB7RP1 in the formulations over the course of Storage time. The determination was carried out using SE-HPLC as described above. The results are shown in Figure 12. Panel A shows the results for storage at 4 ° C. Panel B shows the results for storage at 29 ° C. Ac-hB7RP1 was at least as stable in the self-buffered formulation at 4 ° C as the conventionally buffered formulations. At 29 ° C, the self-buffered formulation was at least as stable as conventionally buffered formulations, and may have been slightly better from 10 weeks to last.

Reference Example 4: pH Stability of Self-buffered Ac-hB7RP1 at 4 ° C and 29 ° C

The self-buffered Ac-hB7RP1 at 60 mg / ml was prepared as described in the Reference Example above. The pH was controlled over time and at the same temperatures as described therein. The results are shown in Figure 13.

Reference Example 5: Buffer Capacity of Ac-hCD22 Formulations- pH 4.0 to 6.0

Self-buffering formulations of Ac-hCD22 were prepared and titrated in the range of pH 5.0 to 4.0 and in the range of 5.0 to 6.0, as described for Ac-hOPGL and Ac-hB7RP1 in the Examples. and Reference Examples above. Buffer capacities were calculated from the titration data, also as described above. The buffer capacity as a function of concentration is shown in Figure 14 for both pH ranges. Panel A shows the buffering capacity of Ac-hCD22 formulations in the range of pH 5.0 to 4.0. Buffering capacity is linearly dependent on concentration, and a formulation of about 21 mg / ml AchCD22 has a buffering capacity equal to that of 10 mM sodium acetate buffer, pH 5.0, measured in the same way. Panel B shows the buffer capacity as a function of concentration in the range of pH 5.0 to 6.0. In this pH range, a formulation of about 30 mg / ml Ac-hCD22 has a buffering capacity equal to that of 10 mM sodium acetate buffer, pH 5.0, measured in the same way.

Reference Example 6: Titration and Buffer Capacities of Ac-hIL4R Formulations - pH 5.0 to 4.0

Self-buffering formulations of Ac-hIL4R were prepared in concentrations of 1, 10, 25 and 90 mg / ml, as described for Ac-hOPGL in the previous Examples. Titrations were carried out using HCl as described above. Figure 15, panel A shows the results of the titrations. Figure 15, Panel B shows the dependence of the buffering capacity on the Ac-hIL4R concentration. The results clearly show the self-buffering ability of Ac-hIL4R in this pH range. At about 75 mg / ml, it provides the same buffering capacity in this pH range as 10 mM sodium acetate buffer, pH 5.0, measured in the same way.

Reference Example 7: Titration and Buffer Capacities of Ac-hIL4R Formulations - pH 5.0 to 6.0

Self-buffering formulations of Ac-hIL4R were prepared in concentrations of 1, 10, 25 and 90 mg / ml, as described for Ac-hOPGL in the previous Examples. Titrations were carried out using NaOH as described above. Figure 16, panel A shows the results of the titrations. Figure 16, Panel B shows the dependence of the buffering capacity of the Ac-hIL4R concentration in this pH range. The results clearly show the self-buffering ability of Ac-hIL4R in this pH range. At about 90 mg / ml, it provides the same buffer capacity in this pH range as 10 mM sodium acetate buffer, pH 5.0, measured in the same way.

Reference Example 8: Stability of Ac-hIL4R and pH in Self-buffered Acetate and Ac-hIL4R Formulations at 37 ° C

Self-buffered and acetate-buffered formulations of Ac-hIL4R were prepared at pH 5.0 and 70 mg / ml as described above. The stability of pH and Ac-hIL4R were monitored in the formulations for 4 weeks at 37 ° C. The stability of Ac-hIL4R was monitored by SE-HPLC as described above. The results are shown in Figure 17. Panel A shows that Ac-hIL4R is at least as stable in the self-buffered formulation as in the sodium acetate buffer formulation. Panel B shows that the pH in the self-buffered formulation is as stable as in the sodium acetate buffer formulation.

Claims (15)

1. A composition of a recombinant antibody and one or more pharmaceutically acceptable polyols, wherein at the pH of the composition, at 21 ° C, at one atmosphere and in equilibrium with the ambient atmosphere, the antibody has a buffer capacity per unit volume of at least that of about 4.0 mM sodium acetate buffer in pure water in the range of pH 5.0 to 4.0 or pH 5.0 to 5.5 under the same conditions, and wherein further, Excluding the buffer capacity of said antibody, the buffer capacity per unit volume of the composition under the same conditions is not more than that of the 2.0 mM sodium acetate buffer in pure water in the range of pH 5.0 to 4 , 0 or pH 5.0 to 5.5 under the same conditions, where the antibody provides at least 80% of the buffer capacity of the composition, where the concentration of the antibody is between about 20 and 400 mg / ml, wherein the pH maintained by the buffering action of the antibody is between approximately 3.5 and 8 , 0, and wherein the antibody comprises: a heavy chain comprising the amino acid sequence of: E VQLLESGGGL VQPGGSLRLS CAASGFTFSS YAMSWVRQAP GKGLEWVSGI TGSGGSTYYA DSVKGRFTIS RDNSKNTLYL QMNSLRAEDT AVYYCAKDPG TTVIMSWFDP WGQGTLVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSNFGTQ

LMISRTPEVT CVVVDVSHED PEVQFNWYVD GVEVHNAKTK PREEQFNSTF RVVSVLTVVH QDWLNGKEYK CKVSNKGLPA PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPMLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK; and a light chain comprising the amino acid sequence of: EIVLTQSPGT LSLSPGERAT LSCRASQSVR GRYLAWYQQK PGQAPRLLIY GASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVFYCQ QYGSSPRTFG QGTKVEIKRT VAAPSVFI FP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC. 2. A composition according to claim 1, wherein the pH maintained by the buffering action of the antibody is between about 4 and 6. 3. A composition according to claim 1, further comprising one or more pharmaceutically acceptable salts. 4. A composition according to claim 3, wherein the total salt concentration is less than 150 mM. 5. A composition according to claim 3, wherein the total salt concentration is less than 100 mM. 6. A composition according to any one of claims 1 to 5, wherein the polyol is one or more of sorbitol, mannitol, sucrose, trehalose or glycerol. 7. A composition according to any one of claims 1 to 6, further comprising one or more pharmaceutically acceptable surfactants. A composition according to claim 7, wherein the surfactant is one or more of polysorbate 20, polysorbate 80, other fatty acid esters of sorbitan, polyethoxylates and poloxamer 188. 9. A composition according to any one of claims 1 to 8, wherein the antibody comprises a first binding moiety of a pair of cognate binding moieties. The composition of any one of claims 1 to 9, wherein the antibody is specific for OPGL. 11. A lyophilizate which upon reconstitution provides a composition according to any of the preceding claims. 12. A kit comprising in one or more packages, a composition according to any one of claims 1 to 10 and instructions with respect to the use thereof or a lyophilisate according to claim 11 and instructions with respect to the use thereof . 13. A composition according to any one of claims 1 to 10 for use in medicine. A process for preparing a composition according to any one of claims 1 to 10, which comprises removing residual buffer using a counter ion or by diafiltration against a buffer-free solution having a pH below the desired pH. A process for preparing a composition according to claim 14, which comprises removing residual buffer using any one or more of the following in the presence of a counter ion: size exclusion chromatography, dialysis and / or tangential flow filtration and / or ion exchange chromatography.